Team:Kyoto/Secretion/Notebook

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[[Image:Header_Kyoto_not_home.jpg|975px]]
-
{{Kyoto/css}}
+
{{Kyoto/header}}  
-
{{Kyoto/header}}
+
= Secretion Notebook =  
-
= Secretion Notebook =
+
<div class="_kyoto-note">  
-
<div class="_kyoto-note">
+
==February 7==  
-
==February 7==
+
====Preculture====  
-
====Preculture====
+
-
<small>by_???</small><br>
+
We started preculture at 12:10.<br>
We started preculture at 12:10.<br>
-
==February 20==
+
==February 8==  
-
==February 21==
+
====Culture====
-
==February 22==
+
We start culturing with 300[mL] of LB medium.<br>  
-
'''PCR'''<br>
+
{|class="wikitable"  
-
{|class="wikitable"
+
!time||OD600
-
! ||Buffer||dNTPs||MgSO4||prefix primer-f||suffix primer-r||product of PCR purification(1ng/μL)||KOD plus||MilliQ||total
+
|-  
-
|-
+
|12:00||start
-
|1||5||5||3||1.5||1.5||0.5||1||32.5||50
+
|-  
-
|-
+
|14:10||0.019
-
|2||5||5||3||1.5||1.5||1||1||32||50
+
|-  
-
|-
+
|14:45||0.154
-
|3||5||5||3||1.5||1.5||2||1||31||50
+
|-  
-
|-
+
|15:05||0.267
-
|4||5||5||3||1.5||1.5||3||1||30||50
+
|-  
-
|-
+
|15:21||0.64
-
|5||5||5||3||1.5||1.5||10||1||29||50
+
|}  
-
|-
+
====Making Competent Cell====
-
|6||5||5||3||1.5||1.5||0||1||33||50
+
We made competent cells.<br><br>  
-
|}
+
====Transformation====
-
94℃, 2min<br>
+
pGEM_TAP<br>LacP (BBa_R0011)<br>DT (BBa_B0015)<br><br>  
-
98℃, 10sec<br>
+
====Making Culture Medium Plates====
-
59℃, 30sec<br>
+
We made 200mL of ampicillin culture, kanamycin culture, and chloramphenicol culture.<br><br>  
-
68℃, 3min<br>
+
====Transformation====
-
→25cycles<br><br>
+
GFP(BBa_E0040) in pSB1A2<br>DT(BBa_B0015) in pSB1AK3<br>ara(BBa_I0500) in pSB2K3<br>LacP(BBa_R0011) in pSB1A2
-
'''Electrophoresis'''<br>
+
-
[[File:Electrophoresis022201.JPG|300px|thumb|right]]
+
-
'''Checking Dpn1'''<br>
+
-
{|class="wikitable"
+
-
!Buffer||lacp(28.7ng/μL)||Dpn1||MilliQ||total
+
-
|-
+
-
|2||10||0.5||7.5||20
+
-
|-
+
-
|2||10||0||5||17
+
-
|}
+
-
'''Gel extraction'''<br>
+
-
lane 1 of the gel 45.0μg/mL<br><br>
+
-
'''PCR purification'''<br>
+
-
product  38.2μg/mL <br><br>
+
-
'''PCR'''<br>
+
-
TMAO reductase<br>
+
-
{|class="wikitable"
+
-
! ||Buffer||dNTPs||MgSO4||prefix primer-f||suffix primer-r||product of gel extract||product of PCR purification(1ng/μL)||KOD plus||MilliQ||total
+
-
|-
+
-
|1||5||5||4||1.5||1.5||0.5||0||1||32.5||50
+
-
|-
+
-
|2||5||5||4||1.5||1.5||1||0||1||32.5||50
+
-
|-
+
-
|3||5||5||4||1.5||1.5||0||0.5||1||32.5||50
+
-
|-
+
-
|4||5||5||4||1.5||1.5||0||1||1||32.5||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
59℃, 30sec<br>
+
-
68℃, 3min<br>
+
-
→25cycles<br><br>
+
-
'''Electrophoresis'''
+
-
[[File:Electrophoresis022202.JPG|300px|thumb|right]]
+
-
1. 1kb ladder<br>
+
-
2. TAMO1<br>
+
-
3. TAMO2<br>
+
-
4. TAMO3<br>
+
-
5. TAMO4<br>
+
-
6. constructive promoter 1-18C<br>
+
-
7. constructive promoter Spe1<br>
+
-
8. constructive promoter Pst1<br>
+
-
9. 1kb ladder<br>
+
-
==February 23==
+
==February 9==  
-
'''Colony PCR'''<br>
+
====transformation====
-
tatABCD(2 samples)<br>
+
BBa-E0040(GFP)(Mr.Fujita)<br><br>  
-
{|class="wikitable"
+
====Liquid culture====
-
!Buffer||dNTPs||MgSO4||primer-f||primer-r||KOD plus||MilliQ||total
+
DT.leap colony transformed on February 8<br>
-
|-
+
colony of competent cell made on February 8<br>
-
|5||5||3||1.5||1.5||1||33||50
+
-
|}
+
-
Predenature 94℃  1min<br>
+
==February 10==
-
Denature    98℃ 10sec<br>
+
====Miniprep====
-
Annealing  59℃ 30sec<br>
+
DT2 43.9μg/ml(1.34 260/230 1.74 260/280)<br>  
-
Extension  68℃ 3min<br>
+
LacP1 17.1μg/ml(1.54 260/280 0.83 260/230)<br>  
-
→25cycles<br><br>
+
LacP2 18.0μg/ml(1.58 260/280 0.87 260/230)<br><br>  
-
'''Electrophoresis'''<br>
+
====transformation====
-
[[File:Electrophoresis022301.JPG|300px|thumb|right]]
+
B0040 1.4k PsB1A2 B0034 1.2M pSB1A2(from iGEM parts plate)<br><br>  
-
1. 1kb ladder 2μL<br>
+
====Competent cell====
-
2. tatABCD 1  5μL + 6×Loading Buffer  1μL<br>
+
We did preculture for overnight. We put 1.5mL of preculture on 150mL of LB culture.
-
3. tatABCD 2  5μL + 6×Loading Buffer  1μL<br>
+
{|class="wikitable"  
-
4. 1kb ladder 2μL<br><br>
+
!time||OD600
-
'''Miniprep'''<br>
+
|-
-
pSB4K5 and pSB3C5<br>
+
|11:45||start
-
deluted it to 25 times and then measured it<br>
+
|-
-
pSB4K5 1 : 60.0 μg/ml       1.67(260/280)      1.98(260/230) <br>
+
|13:30||0.048
-
pSB4K5 2 : 55.0 μg/ml      1.49(260/280)      1.62(260/230) <br>
+
|-  
-
pSB3C5-3 : 3.6 μg/ml       1.57(260/280)      3.00(260/230) <br>    
+
|14:30||0.168
-
pSB3C5-4 : 1.3 μg/ml       1.44(260/280)      1.04(260/230) <br><br>
+
|-
-
'''Liquid culture'''<br>
+
|15:03||0.256
-
pSB3C5-3,4<br><br>
+
|-
-
'''Electrophoresis'''<br>
+
|15:20||0.405
-
[[File:Electrophoresis022302.JPG|300px|thumb|right]]
+
|-
-
1. 1kb ladder 2μL<br>
+
|15:35||0.459
-
2. pSB3C5-3 5μL, 6×loading dye 1μL<br>
+
|-
-
3. pSB3C5-4 5μL, 6×loading dye 1μL<br>
+
|at last||0.576
-
4. 1kb ladder 2μL<br>
+
-
 
+
-
==February 27==
+
-
'''Test of Dpn1'''<br>
+
-
{| class="wikitable"
+
-
!Buffer2||GFP2||BSA||MilliQ||Dpn1
+
-
|-
+
-
|3||3||0.3||23||1
+
|}
|}
-
'''Colony PCR'''<br>
 
-
{| class="wikitable"
 
-
! ||buffer||dNTPs||MgSO4||Primer-f||Primer-r||MilliQ||KOD Plus||total
 
-
|-
 
-
|Colony PCR(2 samples)||5||5||3||1.5||1.5||33||1||50
 
-
|-
 
-
|Negative control||5||5||3||1.5||1.5||34||0||50
 
-
|}
 
-
Predenature 94℃,2min<br>
 
-
Denature 98℃,10sec<br>
 
-
Annealing 59℃,30sec<br>
 
-
Extension 68℃,3min<br>
 
-
→25cycles<br><br>
 
-
'''Electrophoresis'''<br>
 
-
{| class="wikitable"
 
-
! ||sample||Loading Dye||MilliQ
 
-
|-
 
-
|1.1kb ladder||2||0||0
 
-
|-
 
-
|2.product of PCR1||5||1||0
 
-
|-
 
-
|3.product of PCR2||5||1||0
 
-
|-
 
-
|4.product of PCR(Negative control)||5||1||0
 
-
|-
 
-
|5.product of PCR(2/23)||5||1||0
 
-
|-
 
-
|6.GFP2(DPN1)||10||2||0
 
-
|-
 
-
|7.GFP2||3||2||7
 
-
|-
 
-
|8.1kb ladder||2||0||0
 
-
|}
 
-
'''Results of liquid culture'''<br>
 
-
We measure this after dilute it to 10 times.<br>
 
-
{|class="wikitable"
 
-
!pSB3C5-5||pSB3C5-6||pSB3C5-5(1% glucose)||pSB3C5-6(1% glucose)
 
-
|-
 
-
|8.5[µg/ml]||-1.8||-17.9||-18.2
 
-
|}
 
-
'''PCR'''<br>
 
-
{| class="wikitable"
 
-
! ||buffer||dNTPs||MgSO4||Primer-f(prefix)||Primer-r(suffix)||PCR purification product(1ng/µL)||MilliQ||KOD Plus||total
 
-
|-
 
-
|1||5||5||3||1.5||1.5||0.2||32.8||1||50
 
-
|-
 
-
|2||5||5||3||1.5||1.5||0.5||32.5||1||50
 
-
|}
 
-
*PCR purification product was that purification product(75ng/µL) of electrophoresis-3 deluted to 1ng/µL<br>
 
-
Predenature 94℃,2min<br>
 
-
Denature 98℃,10sec<br>
 
-
Annealing 59℃,30sec<br>
 
-
Extension 68℃,3min<br>
 
-
→25cycles<br><br>
 
-
==February 28==
+
==February 11==  
-
'''Electrophoresis'''<br>
+
====Checking Transformation efficiency====
-
1. 1kb ladder<br>
+
Conpetent cell's transformation efficiency is 1.3x10^4colonys/μg<br><br>
-
2. PCR1 →Product of gel extraction : tatABCD with prefix and suffix 105[ng/µL]<br>
+
-
3. PCR2<br><br>
+
-
'''Restriction'''<br>
+
-
{| class="wikitable"
+
-
!Buffer2||plasmid(?)||enzyme||MilliQ||total
+
-
|-
+
-
|2||2||0.2||15.8||20
+
-
|}
+
-
incubate 1 hour at 37℃<br><br>
+
-
'''Electrophoresis'''<br>
+
-
1. 1kb ladder<br>
+
-
2. Control (without enzymes)<br>
+
-
3. EcoR1<br>
+
-
4. Xba1 (crystallized)<br>
+
-
5. Xba1 (with seal)<br>
+
-
6. Spe1<br>
+
-
7. Pst1<br>
+
-
8. 1kb ladder<br><br>
+
-
'''PCR and Electrophoresis'''<br>
+
-
{| class="wikitable"
+
-
!Quick Taq Dye Mix||primer-f||primer-r||template||MilliQ||total
+
-
|-
+
-
|25||1.0||1.0||0.5||22.5||50
+
-
|}
+
-
Predenature 94℃,2min<br>
+
-
Denature 94℃,30sec<br>
+
-
Annealing 59℃,30sec<br>
+
-
Extension 68℃,3min<br>
+
-
→25cycles<br><br>
+
-
'''Restriction'''<br>
+
-
{| class="wikitable"
+
-
!BufferH||tatABCD||EcoR1||Spe1||MilliQ||total
+
-
|-
+
-
|2||5||0.2||0.2||12.6||20
+
-
|}
+
-
'''PCR purification'''<br>
+
-
We eluted the product for 30µL MilliQ<br><br>
+
-
'''Ligation'''<br>
+
-
{| class="wikitable"
+
-
!Insert(tatABCD)||Vector(pSB1C3)||Ligation High||total
+
-
|-
+
-
|10||1||5||16
+
-
|}
+
-
4℃, overnight
+
-
==February 29==
+
==February 13==  
-
'''Transformation'''<br>
+
====Transformation====
-
{|class="wikitable"
+
Const promoter J23110, J23109, J23100<br>  
-
!tatABCD||competent cell||total
+
{|class="wikitable"  
-
|-
+
!DNA||Competent cell||total  
-
|1||10||11
+
|-  
-
|}
+
|1μL||20||21
-
'''Checking Restriction enzyme'''<br>
+
|}  
-
{|class="wikitable"
+
No colony was there on February 14<br><br>  
-
!plasmid seems to be 1-18C promoter||Enzyme||Buffer||MilliQ||total
+
====Liquid culture====
-
|-
+
LacP, DT, RBS(BBa_B0034),GFP<br>  
-
|2||0.2||2||15.8||20
+
start at 20:00<br>  
-
|}
+
in Plus grow with Ampicilin 3mL<br>
-
'''Checking tatABCD'''<br>
+
-
{| class="wikitable"
+
-
!tatABCD||Hind3||Buffer||MilliQ||total
+
-
|-
+
-
|5||0.2||2||12.8||20
+
-
|}
+
-
'''PCR'''<br>
+
-
{| class="wikitable"
+
-
! ||buffer||dNTPs||MgSO4||Primer-f||Primer-r||ColE1(6.5ng/µL) / TMAO||MilliQ||KOD Plus Neo||total
+
-
|-
+
-
|Kil||2.5||2.5||1.5||0.75||0.75||0.5||16||0.5||25
+
-
|-
+
-
|TMAO||2.5||2.5||1.5||0.75||0.75||0.5||16||0.5||25
+
-
|}
+
-
Predenature 94℃,2min<br>
+
-
Denature 98℃,10sec<br>
+
-
Annealing 60℃,30sec<br>
+
-
Extension 68℃,3min<br>
+
-
→30cycles<br><br>
+
-
'''Electrophoresis'''<br>
+
-
1. 1kb ladder<br>
+
-
2. Kil (649bp)<br>
+
-
3. TMAO (2720bp)<br>
+
-
4. TMAO (Quick Taq)<br>
+
-
5. tatABCD (Quick Taq)<br>
+
-
6. tatABCD (Hind3)<br>
+
-
7. 1kb ladder<br>
+
-
==March 1==
+
==February 14==  
-
'''PCR'''<br>
+
-
*TMAO<br>
+
-
Template is gDNA and product of colony PCR gel extraction<br>
+
-
{| class="wikitable"
+
-
! ||Buffer||gNTPs||MgSO4||Primer-f||Primer-r||KOD Plus Neo||Template gDNA||product of gel extraction||DW||total
+
-
|-
+
-
|1||2.5||2.5||1.5||0.75||0.75||0.5||0.5||0||16||25
+
-
|-
+
-
|2||2.5||2.5||1.5||0.75||0.75||0.5||0||2||14.5||25
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 1.5min<br>
+
-
→25cycles<br><br>
+
-
*Kil
+
-
{| class="wikitable"
+
-
!Buffer||dNTPs||MgSO4||primer-f||primer-r||colE1(6.5ng/µL)||KOD Plus Neo||MilliQ||total
+
-
|-
+
-
|2.5||2.5||1.5||0.75||0.75||0.5||16||0.5||25
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
61℃, 30sec<br>
+
-
68℃, 1min<br>
+
-
→20cycles<br><br>
+
-
'''Electrophoresis'''<br>
+
-
1. 1kb ladder<br>
+
-
2. TMAO1 (gDNA)<br>
+
-
3. TMAO2 (product of gel extraction)<br>
+
-
4. Kil<br><br>
+
-
'''PCR'''<br>
+
-
{| class="wikitable"
+
-
!Buffer||dNTPs||MgSO4||primer-f||primer-r||Product of Purification||KOD Plus Neo||MilliQ||total
+
-
|-
+
-
|5||5||3||1.5||1.5||1||32||1||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
61℃, 30sec<br>
+
-
68℃, 30sec<br>
+
-
20cycles<br>
+
-
→Purification 230ng/µL<br><br>
+
-
'''Restriction'''<br>
+
-
{| class="wikitable"
+
-
!Kil||EcoR1||Spe1||BufferH||MilliQ||total
+
-
|-
+
-
|5||0.2||0.2||2||12.6||20
+
-
|}
+
-
incubate at 37℃, for 1.5 hours<br><br>
+
-
'''PCR Purification'''<br><br>
+
-
'''Ligation'''<br>
+
-
{| class="wikitable"
+
-
!Kil||pSB1C3||Ligation High||total
+
-
|-
+
-
|5||1||3||9
+
-
|}
+
-
at 4℃, for overnight
+
-
 
+
-
==March 2==
+
-
'''Ligation'''<br>
+
-
{| class="wikitable"
+
-
!Kil||pSB1C3||Ligation High Ver.2||total
+
-
|-
+
-
|5||1||3||9
+
-
|}
+
-
{| class="wikitable"
+
-
!tatABCD||pSB1C3||Ligation||total
+
-
|-
+
-
|10||1||5||16
+
-
|}
+
-
at 16℃ for 1 hour<br><br>
+
-
'''Transformation'''<br>
+
-
{| class="wikitable"
+
-
!Kil||Kil(3/1,Ligation)||tatABCD||competet cell||total
+
-
|-
+
-
|1||0||0||10||11
+
-
|-
+
-
|0||1||0||10||11
+
-
|-
+
-
|0||0||1||10||11
+
-
|}
+
-
'''PCR'''
+
-
{| class="wikitable"
+
-
!Quick Taq||primer-r||primer-f||template||MilliQ||total
+
-
|-
+
-
|25||1||1||0.5||22.5||50
+
-
|}
+
-
'''Electrophoresis'''
+
-
(27)<br><br>
+
-
'''Restriction'''<br>
+
-
{| class="wikitable"
+
-
!pSB3C5-5||EcoR1||Pst1||BufferH||BSA||MilliQ||total
+
-
|-
+
-
|20||0.5||0.5||3||0.5||5.5||30
+
-
|}
+
-
at 37℃, for 2 hour<br><br>
+
-
'''Electrophoresis'''<br>
+
-
1. 1kb ladder 2µL<br>
+
-
2. pSB3C5 5µL + 6×Loading Buffer 1µL<br>
+
-
・product of gel extraction(about 2700bp)<br>
+
-
－30.9µg/mL<br><br>
+
-
'''Restriction'''<br>
+
-
GFP1,2,3
+
-
{| class="wikitable"
+
-
!GFP||EcoR1||Pst1||Buffer||BSA||MilliQ||total
+
-
|-
+
-
|10||0.5||0.5||3||0.5||15.5||30
+
-
|}
+
-
at 37℃, for 2.5 hours<br><br>
+
-
'''Restriction'''<br>
+
-
{| class="wikitable"
+
-
!DT||EcoR1||Xba1||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|10||0.2||0.2||3||0.3||16.3||30
+
-
|}
+
-
{| class="wikitable"
+
-
!Constitutive Promoter||Spe1||Pst1||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|15||0.2||0.2||3||0.3||11.3||30
+
-
|}
+
-
at 37℃, 2 hours<br>
+
-
*J23117-1:135ng/µL, J23107-1:115ng/µL<br>
+
-
*DT3→PCR Purification<br>
+
-
*Promoter→Gel Extraction<br><br>
+
-
'''Checking TMAO'''<br>
+
-
{| class="wikitable"
+
-
!something seems to be TMAO||Buffer2||EcoR1||MilliQ||total
+
-
|-
+
-
|10||2||0.5||7.5||20
+
-
|}
+
-
at 37℃, for 1 hour<br><br>
+
-
'''Electrophoresis'''<br>
+
-
1. 1kb ladder<br>
+
-
2. GFP1 that had been cut by restriction enzyme<br>
+
-
3. GFP2 that had been cut by restriction enzyme<br>
+
-
4. GFP3 that had been cut by restriction enzyme<br>
+
-
5. GFP1<br>
+
-
6. GFP2<br>
+
-
7. GFP3<br>
+
-
8. TMAO (control)<br>
+
-
9. TMAO (EcoR1)<br>
+
-
10. DT (control)<br>
+
-
11. DT (EcoR1, Xba1)<br>
+
-
12. 1kb ladder<br><br>
+
-
'''Checking tatABCD'''<br>
+
-
{| class="wikitable"
+
-
!Quick Taq||primer-f||primer-r||MilliQ||total
+
-
|-
+
-
|25||1||1|||23||50
+
-
|}
+
-
'''Electrophoresis'''<br>
+
-
(28)
+
-
 
+
-
==March 3==
+
-
====PCR====
+
-
{| class="wikitable" style="text-align: right"
+
-
! ||template||buffer||dNTPs||MgSO4||VF||VR||KOD plus||MilliQ||total
+
-
|-
+
-
|1||1||5||5||3||1.5||1.5||1||32||50
+
-
|-
+
-
|2||2||5||5||3||1.5||1.5||1||31||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
50℃, 30sec<br>
+
-
68℃, 1min<br>
+
-
→30cycles<br><br>
+
====Miniprep====
====Miniprep====
 +
concentration[μg/μL]
 +
{|class="wikitable"
 +
|LacP3||39.6
 +
|-
 +
|LacP4||40.8
 +
|-
 +
|LacP5||28.9
 +
|-
 +
|RBS1||28.2
 +
|-
 +
|RBS2||57.4
 +
|-
 +
|RBS3||13.2
 +
|-
 +
|DT3||69.7
 +
|-
 +
|DT4||64.4
 +
|-
 +
|DT5||61.5
 +
|-
 +
|GFP1||64.0
 +
|-
 +
|GFP2||50.5
 +
|-
 +
|GFP3||66.0
 +
|}
 +
====Restriction====
 +
Const promoterJ23100<br>
 +
{|class="wikitable"
 +
!DNA||Spe1||Pst1||Buffer2||BSA||MilliQ||total
 +
|-
 +
|20||0.5||0.5||3||0.5||5.5||30
 +
|}
 +
at 37℃ for overnight<br>
-
==March 4==
+
==February 15==  
-
====Sequence of tatABCD====
+
====Making gel====
-
{| class="wikitable" style="text-align: right;"
+
1% Agarose gel<br>
-
!Quick Taq||primer-f||promer-r sequence||template||MilliQ||total
+
{|class="wikitable"  
-
|-
+
!Agarose||TAE
-
|25||1||1||1||23||50
+
|-  
-
|}
+
|1.6g||160mL
-
{| class="wikitable" style="text-align: right;"
+
|}
-
!Quick Taq||primer-f sequence||primer-r||template||MilliQ||total
+
====Electrophoresis====
-
|-
+
[[File:Electrophoresis021501.JPG|200px|thumb|right|No.1]]
-
|25||1||1||1||23||50
+
Gel No.1  
-
|}
+
{|class="wikitable"  
-
====Colony PCR of TMAO====
+
!Restriction product||loading dye
-
{| class="wikitable" style="text-align: right;"
+
|-  
-
!buffer||dNTPs||NgSO4||primer-f||primerr-r||KOD plus||MilliQ||total
+
|5μL||1  
-
|-
+
|}
-
|5||5||4||1.5||1.5||1||32||50
+
The marker was 1kb ladder<br>
-
|}
+
It seemed that this restriction product was not cut.<br><br>
-
→ethanol precipitation<br>
+
[[File:Electrophoresis021502.JPG|200px|thumb|right|No.2]]
 +
Gel No.2<br>
 +
Lane1 : 1kb ladder<br>
 +
Lane2 : J23100 2μL + 6*Loading dye 1μL<br>
 +
Lane3 : J23100(Spe1,Pst1) 5μL<br>
 +
Lane4 : J23100(Spe1,Pst1) 2μL<br>
 +
*There were bands on lane_2 and we cannot identify these bands because the sample of lane_2 was not cut with any restriction enzyme.<br>
 +
*There must have been bands at 2100bp and 883bp on lane_3 and lane_4.<br><br>
 +
====Testing whether restriction enzyme were deactivated or not====
 +
{|class="wikitable"  
 +
!DNA(DT)||restriction enzyme||Buffer||BSA||MilliQ||total  
 +
|-  
 +
|10||0.5||3||0.5||16||30
 +
|}  
 +
at 37℃ for Oveernight<br>
 +
Restriction enzyme means Spe1(1,2)  Pst1(1,2,3) in this time.<br><br><br><br><br><br><br><br><br><br><br><br>
-
94℃, 2min<br>
+
==February 16==
-
94℃, 30sec<br>
+
-
55℃, 30sec<br>
+
-
68℃, 2.5min<br>
+
-
→25cycles<br><br>
+
====Electrophoresis====
====Electrophoresis====
-
1. 1kb ladder<br>
+
[[File:Electrophoresis021601.JPG|300px|thumb|right]]
-
2. tatABCD1<br>
+
1. 1kb ladder<br>  
-
3. tatABCD2<br>
+
2. DT2<br>  
-
4. TMAO<br>
+
3. DT3<br>  
-
5. 1kb ladder<br><br>
+
4. DT2 (Spe1-1)<br>  
-
====Restriction====
+
5. DT2 (Spe1-2)<br>
-
{| class="wikitable"
+
6. DT2 (Pst1-1)<br>
-
!TMAO||EcoR1||BufferH||BSA||MilliQ||total
+
7. DT2 (Pst1-2)<br>
-
|-
+
8. DT2 (Pst1-3)<br>
-
|10||0.2||3||0.3||16.5||30
+
9. DT3 (Pst1-4)<br>
-
|}
+
10. 1kb ladder<br><br>
-
{|class="wikitable"
+
Pst1-1, Pst1-2, and Pst1-3 did not cut DNAs. They seemed to be deactivated.<br><br>  
-
!TMAO||Xba1||Pst1||BudderM||BSA||MilliQ||total
+
====Genomic PCR====
-
|-
+
{|class="wikitable"  
-
|10||0.2||0.2||3||0.3||16.3||30
+
!10*Buffer for KOD Plus||2mM dNTPs||25mM MgSO4||10μM primer-f||10μM primer-r||158ng/μL Genomic DNA||KOD plus||MilliQ||total
-
|}
+
|-
-
at 37℃ for 1 hour<br><br>
+
|5||5||3||1.5||1.5||1||1||32||50
 +
|}  
====Electrophoresis====
====Electrophoresis====
-
<br>
+
[[File:Electrophoresis021602.JPG|300px|thumb|right]]
-
 
+
1. 1kb ladder<br>  
 +
2. tatABCD (2.5kb)<br>
 +
3. TAMO reductase (2.7kb)<br>
 +
4. Negative control<br>
 +
We got bands of tatABCD but there were nonspecific amplification products.<br>
 +
We failed amplification of TAMO reductase.<br><br><br><br><br><br><br><br>
====Transformation====
====Transformation====
-
{|class="wikitable"
+
Constitutive promoter (BBa_J23107 , BBA_J23117)<br>
-
!pSB1C3||competent cell(made at 2/8)||total
+
High copy plasmid (pSB1AT3)<br>
-
|-
+
{|class="wikitable"  
-
|5||100||105
+
!DNA||competent cell  
 +
|-  
 +
|1μL||10μL
|}
|}
-
==March 5==
+
==February 17==  
-
====Restriction====
+
-
{| class="wikitable"
+
-
!pSB1C3(Xba1, Spe1)||Pst1||BufferH||BSA||MilliQ||total
+
-
|-
+
-
|10||0.2||2||0.2||7.6||20
+
-
|-
+
-
!pSB1C3(Xba1, Spe1)||EcoR1||BufferH||BSA||MilliQ||total
+
-
|-
+
-
|10||0.2||2||0.2||7.6||20
+
-
|}
+
-
at 37℃ for 1 hour<br>
+
-
→Then we did ethanol precipitation<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!Kil(EcoR1, Spe1)||pSB1C3(EcoR1)||Ligation High||total
+
-
|-
+
-
|5||1||3||9
+
-
|}
+
-
at 16℃ for 1 hour<br><br>
+
-
====Transformation====
+
-
{| class="wikitable"
+
-
!Kil||competent cell||total
+
-
|-
+
-
|1||10||11
+
-
|}
+
-
We used commercially available competent cells in this time.<br><br>
+
====PCR====
====PCR====
-
TMAO<br>
+
We did PCR to amplify products of PCR that we had done yesterday but we could not amplify tatABCD.<br><br>
-
{| class="wikitable"
+
====Genomic PCR====
-
!buffer||dNTPs||MgSO4||Primer-f||Primer-r||Template||KOD plus||MilliQ||total
+
{|class="wikitable"  
-
|-
+
!Buffer||dNTPs||MgSO4||primer-f||primer-r||genomic DNA||KOD plus||MilliQ||total  
-
|5||5||3||1.5||1.5||1||1||32||50
+
|-  
-
|}
+
|2.5||2.5||5||0.75||0.75||0.5||0.5||16||50  
 +
|}  
 +
Predenature  94℃,  2min<br>
 +
Denature      98℃,  10sec<br>
 +
Annealing    57℃,  30sec<br>
 +
Extension    68℃, 2.5min<br>
 +
(30cycles)<br><br>
====Electrophoresis====
====Electrophoresis====
-
(31)<br><br>
+
[[File:Electrophoresis021701.JPG|300px|thumb|right]]
 +
1. 1kb ladder<br>
 +
2. TAMO reductase<br>
 +
3. Negative control<br><br>  
====Restriction====
====Restriction====
-
{| class="wikitable"
+
{|class="wikitable"  
-
! ||Lacp||pSB3C5||EcoR1||Pst1||BufferH||BSA||MilliQ||total
+
!J23100||Spe1||Pst1||Buffer2||BSA||MilliQ||total
-
|-
+
|-
-
|1||20||0||0.5||0.5||3||0.5||5.5||30
+
|10||0.5||0.5||3||0.5||15.5||30
-
|-
+
|}
-
|2||0||20||0.5||0.5||3||0.5||5.5||30
+
====Genomic PCR====
-
|}
+
{|class="wikitable"
-
at 37℃ for 1 hour<br>
+
! ||Buffer||dNTPs||MgSO4||primer-f||primer-r||genomic DNA||KOD plus||MilliQ||total  
-
1→Ethanol precipitation　45.4µg/mL<br>
+
|-  
-
2→Gel extraction　38.7µg/mL<br><br>
+
|TMAO reductase||2.5||2.5||3||0.75||0.75||0.5||0.5||15.5||25
-
====Ligation====
+
|-
-
{| class="wikitable"
+
|tatABCD||2.5||2.5||2||0.75||0.75||0.5||0.5||15.5||25
-
!LacP||pSB3C5||Ligation High||total
+
|-  
-
|-
+
|tatABCD||2.5||2.5||2||0.75||0.75||5||0.5||10.5||25
-
|10||2||6||18
+
|}
-
|}
+
====Electrophoresis====
-
at 4℃ for overnight<br><br>
+
[[File:Electrophoresis021702.JPG|300px|thumb|right]]
 +
1. 1kb ladder <br>
 +
2.3.  TAMO reductase<br>
 +
4. tatABCD<br>
 +
5. tatABCD(10 times amount of genome)<br><br>
 +
'''Checking of restriction enzyme'''<br>
 +
{|class="wikitable"
 +
!DT||enzyme||Buffer||BSA||MilliQ||total
 +
|-
 +
|2||0.5||3||0.5||24||30  
 +
|}  
 +
at 37℃ for overnight<br>  
 +
We checked EcoR1 and Xba1.<br><br><br><br><br><br><br><br>
 +
 
 +
==February 18==
 +
====Miniprep====
 +
{|class="wikitable"  
 +
! ||μg/mL||260/280||230/260
 +
|-  
 +
|JS3117-1||135||1.5||2.06
 +
|-
 +
|JS3117-2||75||1.6||1.63
 +
|-
 +
|JS3109-1||115||1.5||1.88
 +
|-
 +
|JS3109-2||75||1.65||1.71
 +
|-
 +
|pSB1AT3-1||70||1.66||1.83
 +
|-
 +
|pSB1AT3-2||100||1.52||1.54
 +
|}  
 +
diluted to 25 times<br><br>  
 +
====Competent cell====
 +
We put 3mL of preculture product on yesterday onto 300mL of LB medium
 +
{|class="wikitable"
 +
!time||OD600
 +
|-
 +
|10:30||start
 +
|-
 +
|12:10||0.118
 +
|-
 +
|13:00||0.270
 +
|-
 +
|13:30||0.502
 +
|}
====Transformation====
====Transformation====
-
{| class="wikitable"
+
{|class="wikitable"  
-
!Lacp+pSB3C5||competent cell||total
+
!pSB1AT3-2||competent cell||MilliQ||total  
-
|-
+
|-  
-
|1||10||11
+
|0||20||10||30
-
|}
+
|-
-
on ice for 30 mins.<br>
+
|2||20||8||30
-
heat shock at 42℃ for 60secs<br>
+
|-
-
on ice for 2 mins.<br>
+
|10||20||0||30
-
After we incubate with 200µL of SOC culture for 1 hour, we did plating on LB culture with CP<br><br>
+
|}  
 +
*Results(on Feb. 19)<br>  
 +
{|class="wikitable"
 +
!pSB1AT3-2||number of colony
 +
|-
 +
|0||0
 +
|-
 +
|2||177
 +
|-
 +
|10||590
 +
|}
 +
Transformation efficiency      7.4x10^4 colonys/μg<br>
 +
 
 +
==February 20==
====Restriction====
====Restriction====
-
{| class="wikitable"
+
sample 1<br>
-
!GFP Plasmid||EcoR1||Spe1||Buffer2||BSA||MilliQ||total
+
{|class="wikitable"
-
|-
+
!DT plasmid||EcoR1||Xba1||Buffer||BSA||MilliQ||total
-
|10||0.5||0.5||3||0.5||15.5||30
+
|-
-
|}
+
|7.5||0.5||0.5||3||0.5||18||30
-
at 37℃ for 2 hours
+
|}
 +
sample2<br>
 +
{|class="wikitable"  
 +
!GFP plasmid||EcoR1||Spe1||Buffer||BSA||MilliQ||total  
 +
|-  
 +
|10||0.5||0.5||3||0.5||15.5||30  
 +
|}  
====Electrophoresis====
====Electrophoresis====
-
1. Ladder 2µL<br>
+
*sample1<br>
-
2. GFP Plasmid (already restricted) 2µL + Loading Dye 2µL + MilliQ 8µL<br>
+
[[File:Electrophoresis022001_.JPG|200px|thumb|right]]
-
3. Ladder 2µL<br>
+
a. sample1 2μL + MilliQ 3μL + 6×Loading Dye 1μL<br>  
 +
b. sample1 5μL + 6×Loading Dye 1μL<br>
 +
lane_1 1kb ladder<br>
 +
lane_2 a<br>
 +
lane_3 b<br>
 +
lane_4 a<br>
 +
lane_5 b<br>
 +
lane_6 a<br>
 +
lane_7 b<br>
 +
lane_8 1kb ladder<br>
 +
*sample2<br>
 +
[[File:Electrophoresis022302.JPG|200px|thumb|right]]
 +
c. sample2 2μL + MilliQ 3μL + 6×Loading Dye 1μL<br>  
 +
d. sample2 5μL + 6×Loading Dye 1μL<br>
 +
lane_1 1kb ladder<br>
 +
lane_2 c<br>
 +
lane_3 d<br>
 +
lane_4 1kb ladder<br><br>  
====PCR====
====PCR====
-
torA signal and pspA
+
{|class="wikitable"
-
pspAはコロニーPCR<br>
+
! ||Buffer||dNTPs||MgSO4||primer-f||primer-r||genomic DNA||KOD plus||MilliQ||total
-
{| class="wikitable"
+
|-
-
!Buffer||dNTPs||MgSO4||Primer-f||Primer-r||template(TMAO)||KOD plus||MilliQ||total
+
|tatABCD1||2.5||2.5||1.5||0.75||0.75||0.5||0.5||16||25
-
|-
+
|-
-
|2.5||2.5||1.5||0.75||0.75||0.5||0.5||16||25
+
|tatABCD2||2.5||2.5||1.5||0.75||0.75||0.5||0.5||16||25
-
|}
+
|-
-
94℃, 2min<br>
+
|TMAO reductase1||2.5||2.5||2||0.75||0.75||0.5||0.5||15.5||25
-
98℃, 10sec<br>
+
|-
-
60℃, 30sec<br>
+
|TMAO reductase2||2.5||2.5||2||0.75||0.75||5||0.5||10.5||25
-
68℃, 30sec<br>
+
|}
-
====electrophoresis====
+
Predenature  94℃    2min<br>
-
1. 100bp Ladder<br>
+
Denature      98℃    10sec<br>
-
2. torA signal (272bp)<br>
+
Annealing    59℃    30sec<br>
-
3. pspA (969bp)<br>
+
Extension    68℃    2.5min<br>
-
4. 100bp Ladder<br>
+
→30cycles<br><br>  
 +
{|class="wikitable"  
 +
! ||Buffer||dNTPs||MgSO4||primer-f||primer-r||PCR products||genomic DNA||KOD plus||MilliQ||total  
 +
|-  
 +
|tatABCD1||2.5||2.5||1.5||0.75||0.75||0||0.5||0.5||16||25
 +
|-
 +
|tatABCD2||2.5||2.5||1.5||0.75||0.75||1||0||0.5||15.5||25
 +
|-
 +
|TMAO reductase1||2.5||2.5||2||0.75||0.75||0||0||0.5||16||25
 +
|-
 +
|TMAO reductase2||2.5||2.5||2||0.75||0.75||0||0||0.5||16||25  
 +
|}  
 +
Predenature  94℃   2min<br>  
 +
Denature    98℃   10sec<br>  
 +
Annealing    59℃    30sec<br>  
 +
Extension    68℃ 2.5min<br>  
 +
→35cycles<br><br>
 +
'''Electrophoresis'''<br>
 +
[[File:Electrophoresis022003.JPG|200px|thumb|right]]
 +
lane_1.1kb ladder<br>  
 +
lane_2.tatABCD<br>  
 +
lane_3.tatABCD<br>  
 +
lane_4.TAMO1<br>
 +
lane_5.TAMO2<br>
 +
lane_6.1kb ladder<br><br><br><br><br>
 +
[[File:Electrophoresis022004.JPG|200px|thumb|right]]
 +
lane_1.1kb ladder<br>
 +
lane_2.tatABCD1<br>
 +
lane_3.tatABCD2<br>
 +
lane_4.TAMO<br>
 +
lane_5.TAMO<br>
 +
lane_6.1kb ladder<br><br><br><br><br><br>
-
==March 6==
+
==February 21==  
-
====Restriction====
+
====PCR (Advantage HF protocol)====
-
{| class="wikitable"
+
{|class="wikitable"  
-
!GFP||EcoR1||Spe1||BufferM||BSA||MilliQ||total
+
! ||buffer||dNTPs||primer-f||primer-r||gDNA||PCR products||DW||polymerase||total  
-
|-
+
|-  
-
|12||0.5||0.5||3||0.5||13.5||30
+
|tatABCD||2.5||2.5||0.75||0.75||1||0||17||0.5||25
-
|-
+
|-  
-
|}
+
|TMAO||2.5||2.5||0.75||0.75||0||1||17||0.5||25
-
We did incubate at 37℃ for 1.5hours.<br>
+
|}  
-
And we did gel extraction on 3/7 and get 40.0μg/mL GFP.<br><br>
+
Predenature  94℃    1min<br>
 +
Denature    94℃    30sec<br>
 +
Annealing    58℃    30sec<br>
 +
Extension    68℃    3min<br>
 +
→25cycles<br><br>
 +
====Electrophoresis====
 +
[[File:Electrophoresis0221.JPG|250px|thumb|right]]
 +
1. 1kb ladder  2μL<br>
 +
2. tatABCD  5μL + 6×Loading Buffer  1μL<br>  
 +
3. TAMO  5μL + 6×Loading Buffer  1μL<br>
 +
4. 1kb ladder  2μL<br><br>
 +
'''Liquid culture'''<br>
 +
pSB3C5-1,2<br>
 +
pSB4K5-1,2
 +
 
 +
==February 22==
 +
====Gel extraction====
 +
lane 1 of the gel 45.0μg/mL<br><br>
 +
====PCR purification====
 +
product  38.2μg/mL <br><br>  
====PCR====
====PCR====
-
{| class="wikitable"
+
TMAO reductase<br>
-
!buffer||dNTPs||MgSO4||Primer-f||Primer-r||template||KOD plus neo||MilliQ||total
+
{|class="wikitable"  
-
|-
+
! ||Buffer||dNTPs||MgSO4||prefix primer-f||suffix primer-r||product of gel extract||product of PCR purification(1ng/μL)||KOD plus||MilliQ||total  
-
|5||5||3||1.5||1.5||0.5||1||32.5||50
+
|-  
 +
|1||5||5||4||1.5||1.5||0.5||0||1||32.5||50
 +
|-
 +
|2||5||5||4||1.5||1.5||1||0||1||32.5||50
 +
|-
 +
|3||5||5||4||1.5||1.5||0||0.5||1||32.5||50  
 +
|-
 +
|4||5||5||4||1.5||1.5||0||1||1||32.5||50
 +
|}
 +
94℃, 2min<br>
 +
98℃, 10sec<br>
 +
59℃, 30sec<br>
 +
68℃, 3min<br>
 +
→25cycles<br><br>
 +
====Electrophoresis====
 +
[[File:Electrophoresis022201.JPG|250px|thumb|right]]
 +
1. 1kb ladder<br>
 +
2. TAMO1<br>
 +
3. TAMO2<br>
 +
4. TAMO3<br>
 +
5. TAMO4<br>
 +
6. constructive promoter 1-18C<br>
 +
7. constructive promoter Spe1<br>
 +
8. constructive promoter Pst1<br>
 +
9. 1kb ladder<br>
 +
====PCR====
 +
{|class="wikitable"
 +
! ||Buffer||dNTPs||MgSO4||prefix primer-f||suffix primer-r||product of PCR purification(1ng/μL)||KOD plus||MilliQ||total
 +
|-
 +
|1||5||5||3||1.5||1.5||0.5||1||32.5||50
 +
|-
 +
|2||5||5||3||1.5||1.5||1||1||32||50
 +
|-
 +
|3||5||5||3||1.5||1.5||2||1||31||50
 +
|-
 +
|4||5||5||3||1.5||1.5||3||1||30||50
 +
|-
 +
|5||5||5||3||1.5||1.5||10||1||29||50
 +
|-
 +
|6||5||5||3||1.5||1.5||0||1||33||50
 +
|}
 +
94℃, 2min<br>
 +
98℃, 10sec<br>
 +
59℃, 30sec<br>
 +
68℃, 3min<br>
 +
→25cycles<br><br>
 +
====Electrophoresis====
 +
[[File:Electrophoresis022202.JPG|300px|thumb|right]]<br><br><br><br><br><br><br><br><br><br><br><br>
 +
====Checking Dpn1====
 +
{|class="wikitable"
 +
!Buffer||LacP(28.7ng/μL)||Dpn1||MilliQ||total
 +
|-
 +
|2||10||0.5||7.5||20
 +
|-
 +
|2||10||0||5||17
|}
|}
-
94℃　2min<br>
 
-
98℃　10sec<br>
 
-
60℃　30sec<br>
 
-
68℃　(torA 10sec / pspA 30sec)    25 cycles<br>
 
-
We used product of PCR on 3/5 of torA and pspA as template.<br><br>
 
-
====Restriction====
 
-
{| class="wikitable"
 
-
!Kil||EcoR1||Spe1||BufferM||BSA||MilliQ||total
 
-
|-
 
-
|10||0.3||0.3||3||0.3||16.1||30
 
-
|}
 
-
{| class="wikitable"
 
-
!DT||EcoR1||Xba1||Buffer2||BSA||MilliQ||total
 
-
|-
 
-
|10||0.5||0.5||3||0.5||15.5||30
 
-
|}
 
-
at 37℃ for 2 hours<br>
 
-
→ purification 37.7ng/μL<br><br>
 
-
====Ligation====
 
-
{| class="wikitable"
 
-
!Kil||pSB1C3||Ligation High Ver.2||total
 
-
|-
 
-
|4||2||3||9
 
-
|}
 
-
*Kil : 350fmol<br>
 
-
*pSB1C3 : 29fmol<br>
 
-
at 16℃ for overnight<br>
 
-
==March 7==
+
==February 23==  
 +
====Colony PCR====
 +
tatABCD(2 samples)<br>
 +
{|class="wikitable"
 +
!Buffer||dNTPs||MgSO4||primer-f||primer-r||KOD plus||MilliQ||total
 +
|-
 +
|5||5||3||1.5||1.5||1||33||50
 +
|}
 +
Predenature 94℃  1min<br>
 +
Denature    98℃ 10sec<br>
 +
Annealing  59℃ 30sec<br>
 +
Extension  68℃  3min<br>
 +
→25cycles<br><br>
====Electrophoresis====
====Electrophoresis====
-
[[File:Electrophoresis0307.JPG|400px|thumb|right]]<br>
+
[[File:Electrophoresis022301.JPG|300px|thumb|right]]  
-
1. 1kb ladder 2µL<br>
+
1. 1kb ladder 2μL<br>  
-
2. pspA (PCR product)2.5µL + Loading Dye 0.5µL<br>
+
2. tatABCD 1  5μL + 6×Loading Buffer  1μL<br>  
-
3. GFP 30µL + Loading Dye 6µL<br>
+
3. tatABCD 2  5μL + 6×Loading Buffer  1μL<br>  
-
4. 1kb ladder 2µL<br>
+
4. 1kb ladder 2μL<br><br>  
-
*The GFP was gel extracted on 3/6 and concentrated by Vacuum in 150µg/mL<br><br>
+
====Miniprep====
-
====Ligation====
+
pSB4K5 and pSB3C5<br>  
-
{| class="wikitable"
+
deluted it to 25 times and then measured it<br>  
-
!VectorDNA||GFP||Ligation High Ver.2||total
+
pSB4K5 1 : 60.0 μg/ml      1.67(260/280)      1.98(260/230) <br>  
-
|-
+
pSB4K5 2 : 55.0 μg/ml      1.49(260/280)      1.62(260/230) <br>  
-
|5||15||10||30
+
pSB3C5-3 : 3.6 μg/ml        1.57(260/280)      3.00(260/230) <br>  
-
|}
+
pSB3C5-4 : 1.3 μg/ml        1.44(260/280)      1.04(260/230) <br><br>  
-
====Restriction====
+
-
{| class="wikitable"
+
-
!torA||EcoR1||Spe1||bufferM||BSA||MilliQ||total
+
-
|-
+
-
|10||0.3||0.3||3||0.3||16.1||30
+
-
|}
+
-
{| class="wikitable"
+
-
!pspA||EcoR1||Spe1||bufferM||BSA||MilliQ||total
+
-
|-
+
-
|5||0.3||0.3||3||0.3||21.1||30
+
-
|}
+
-
at 37℃ for 1.5 hours<br><br>
+
-
====Purification====
+
-
torA→31.8ng/µL<br>
+
-
pspA→49.3ng/µL<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!torA||pSB1C3||Ligation High Ver.2||total
+
-
|-
+
-
|3||3||3||9
+
-
|}
+
-
{| class="wikitable"
+
-
!pspA||pSB1C3||Ligation High Ver.2||total
+
-
|-
+
-
|4||2||3||9
+
-
|}
+
-
at 4℃, for overnight<br>
+
-
*torA→31.8ng/µL×3µL=95.4ng=0.529pmol<br>
+
-
*pSB1C3→19.4ng/µL×3µL=58.2ng=0.042pmol<br>
+
-
*pspA→49.3ng/µL×4µL=197.2ng=0.308pmol<br>
+
-
*pSB1C3→19.4ng/µL×2µL=38.8ng=0.029pmol<br>
+
-
 
+
-
==March 8==
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!pSB4K5||EcoR1||Spe1||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|20||0.2||0.2||3||0.2||6.4||30
+
-
|-
+
-
|}
+
-
at 37℃ for 1 hour.<br>
+
-
→Purification : 36.6ng/µL<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!Kil||pSB4K5||Ligation High Ver.2||total
+
-
|-
+
-
|10||1||5||16
+
-
|}
+
-
at 4℃ for overnight<br>
+
-
*Kil→37.7ng/µL×10µL=377ng=879fmol<br>
+
-
*pSB4K5→36.6ng/µL×1µL=36.6ng=86fmol<br><br>
+
====Liquid culture====
====Liquid culture====
-
Lacp + pSB3C5 -1, 2<br><br>
+
pSB3C5-3,4<br><br>  
-
====Transformation====
+
====Electrophoresis====
-
{| class="wikitable"
+
[[File:Electrophoresis022302.JPG|300px|thumb|right]]
-
!torA||pspA||competent cell||total
+
1. 1kb ladder 2μL<br>
-
|-
+
2. pSB3C5-3 5μL, 6×loading dye 1μL<br>
-
|1||0||10||11
+
3. pSB3C5-4 5μL, 6×loading dye 1μL<br>
-
|-
+
4. 1kb ladder 2μL<br><br><br><br><br><br><br><br>
-
|0||1||10||11
+
-
|}
+
-
We use commercially available competent cells in this time.<br>
+
-
==March 9==
+
==February 27==  
-
====Restriction====
+
====Test of Dpn1====
-
{| class="wikitable"
+
{| class="wikitable"  
-
!tatABCD||Xba1||Pst1||BufferM||BSA||MilliQ||total
+
!Buffer2||GFP2||BSA||MilliQ||Dpn1
-
|-
+
|-  
-
|10||0.2||0.2||3||0.3||16.3||30
+
|3||3||0.3||23||1
-
|}
+
|}  
-
at 37℃ for 1hour<br>
+
-
→purification : 75.0μg/mL    (1.11  260/280 , 0.81  260/230)<br><br>
+
-
====Miniprep====
+
-
lacP + pSB3C5 1    80.5μg/mL    (1.78  260/280 , 2.00  260/230)<br>
+
-
lacP + pSB3C5 2    107.2μg/mL  (1.83  260/280 , 1.90  260/230)<br><br>
+
====Colony PCR====
====Colony PCR====
-
{| class="wikitable"
+
{| class="wikitable"  
-
!Quick Taq||VF||VR||MilliQ||total
+
! ||buffer||dNTPs||MgSO4||Primer-f||Primer-r||MilliQ||KOD Plus||total  
 +
|-
 +
|Colony PCR(2 samples)||5||5||3||1.5||1.5||33||1||50
 +
|-
 +
|Negative control||5||5||3||1.5||1.5||34||0||50
 +
|}
 +
Predenature 94℃,2min<br>
 +
Denature 98℃,10sec<br>
 +
Annealing 59℃,30sec<br>
 +
Extension 68℃,3min<br>
 +
→25cycles<br><br>
 +
====Electrophoresis====
 +
[[File:Electrophoresis0227.JPG|200px|thumb|right]]
 +
{| class="wikitable"
 +
! ||sample||Loading Dye||MilliQ
 +
|-
 +
|1.1kb ladder||2||0||0
 +
|-
 +
|2.product of PCR1||5||1||0
 +
|-
 +
|3.product of PCR2||5||1||0
 +
|-
 +
|4.product of PCR(Negative control)||5||1||0
 +
|-
 +
|5.product of PCR(2/23)||5||1||0
 +
|-
 +
|6.GFP2(DPN1)||10||2||0
 +
|-
 +
|7.GFP2||3||2||7
 +
|-
 +
|8.1kb ladder||2||0||0
 +
|}
 +
====Results of liquid culture====
 +
We measure this after dilute it to 10 times.<br>
 +
{|class="wikitable"
 +
!pSB3C5-5||pSB3C5-6||pSB3C5-5(1% glucose)||pSB3C5-6(1% glucose)
|-
|-
-
|25||1||1||23||50
+
|8.5[µg/ml]||-1.8||-17.9||-18.2
-
|}
+
|}  
-
94℃ 2min<br>
+
====PCR====
-
94℃ 30sec<br>
+
{| class="wikitable"  
-
55℃ 30sec<br>
+
! ||buffer||dNTPs||MgSO4||Primer-f(prefix)||Primer-r(suffix)||PCR purification product(1ng/µL)||MilliQ||KOD Plus||total
-
68℃ 6sec<br>
+
|-
-
25cycles<br><br>
+
|1||5||5||3||1.5||1.5||0.2||32.8||1||50
-
====Ligation====
+
|-  
-
{| class="wikitable"
+
|2||5||5||3||1.5||1.5||0.5||32.5||1||50
-
!tatABCD||constP J23107||Ligation High Ver.2||total
+
|}  
-
|-
+
*PCR purification product was that purification product(75ng/µL) of electrophoresis-3 deluted to 1ng/µL<br>
-
|5||1||3||9
+
Predenature 94℃,2min<br>
-
|}
+
Denature 98℃,10sec<br>
-
tatABCD : 227fmol<br>
+
Annealing 59℃,30sec<br>
-
constP J23107 : 21fmol<br><br>
+
Extension 68℃,3min<br>
-
====Transformation====
+
→25cycles<br><br>
-
{| class="wikitable"
+
 
-
! ||pspA||torA||Kil||competent cell
+
==February 28==  
-
|-
+
====Electrophoresis====
-
|1||1||0||0||10
+
[[File:Electrophoresis022801.JPG|250px|thumb|right]]
-
|-
+
1. 1kb ladder<br>  
-
|2||0||1||0||10
+
2. PCR1 →Product of gel extraction : tatABCD with prefix and suffix 105[ng/µL]<br>
-
|-
+
3. PCR2<br><br>  
-
|3||0||0||1||10
+
-
|}
+
-
====Miniprep====
+
-
4mL of plusgrow which had been cultured for overnight.<br>
+
-
pSB4K5 : 80.5μg/mL<br>
+
-
==March 10==
 
-
====Screening PCR====
 
-
{| class="wikitable"
 
-
!Quick Taq||VF||VR||MilliQ||total
 
-
|-
 
-
|25||1||1||23||50
 
-
|}
 
-
[[File:Electrophoresis0310.JPG|400px|thumb|right]]
 
-
Then we did electrophoresis to confirm.<br>
 
-
1.1kb ladder<br>
 
-
2.kil (649bp)<br>
 
-
3,4,5, pspA (969bp)<br>
 
-
6,1kb ladder<br><br>
 
-
1, 100bp ladder<br>
 
-
2,3,4, torA signal<br><br>
 
====Restriction====
====Restriction====
-
{| class="wikitable"
+
{| class="wikitable"  
-
!LacP-pSB3C5||Spe1||Pst1||BufferM||BSA||MilliQ||total
+
!Buffer2||plasmid(?)||enzyme||MilliQ||total  
-
|-
+
|-  
-
|10||0.2||0.2||2||0.2||7.4||20
+
|2||2||0.2||15.8||20
-
|}
+
|}
-
for 2.5 hours at 37℃<br>
+
incubate 1 hour at 37℃<br><br>
-
→purification  29.0ng/μL<br>
+
====Electrophoresis====
-
{| class="wikitable"
+
[[File:Electrophoresis022802.JPG|250px|thumb|right]]
-
!torA||Xba1||Pst1||BufferM||BSA||MilliQ||total
+
[[File:Electrophoresis022803.JPG|250px|thumb|right]]
-
|-
+
1. 1kb ladder<br>
-
|10||0.2||0.2||2||0.2||7.4||20
+
2. Control (without enzymes)<br>
-
|}
+
3. EcoR1<br>
-
for 2.5 hours at 37℃<br>
+
4. Xba1 (crystallized)<br>
-
→purification  91.8ng/μL<br><br>
+
5. Xba1 (with seal)<br>  
 +
6. Spe1<br>  
 +
7. Pst1<br>
 +
8. 1kb ladder<br><br>
 +
====PCR and Electrophoresis====
 +
{| class="wikitable"  
 +
!Quick Taq Dye Mix||primer-f||primer-r||template||MilliQ||total  
 +
|-  
 +
|25||1.0||1.0||0.5||22.5||50
 +
|}
 +
Predenature 94℃,2min<br>
 +
Denature 94℃,30sec<br>
 +
Annealing 59℃,30sec<br>
 +
Extension 68℃,3min<br>
 +
→25cycles<br><br>
 +
====Restriction====
 +
{| class="wikitable"
 +
!BufferH||tatABCD||EcoR1||Spe1||MilliQ||total
 +
|-
 +
|2||5||0.2||0.2||12.6||20  
 +
|}  
 +
====PCR purification====
 +
We eluted the product for 30µL MilliQ<br><br>  
====Ligation====
====Ligation====
-
{| class="wikitable"
+
{| class="wikitable"  
-
!torA||Lacp-pSB3C5||Ligation High Ver.2||total
+
!Insert(tatABCD)||Vector(pSB1C3)||Ligation High||total  
-
|-
+
|-  
-
|4||2||3||9
+
|10||1||5||16
-
|}
+
|}  
-
torA : 929fmol<br>
+
4℃, overnight
-
Lacp-pSB3C5 : 284fmol<br>
+
-
for overnight at 4℃
+
-
==March 11==
+
==February 29==  
-
====Miniprep====
+
====Transformation====
-
We used 3μL of plus grow that we had cultured for overnight.<br>
+
{|class="wikitable"
-
torA : 62.8ng/μL<br><br>
+
!tatABCD||competent cell||total
-
====Screening PCR====
+
|-
-
{| class="wikitable"
+
|1||10||11
-
!Quick Taq||VF||VR||MilliQ||total
+
|}
-
|-
+
====Checking Restriction enzyme====
-
|25||1||1||23||50
+
{|class="wikitable"
-
|}
+
!plasmid seems to be 1-18C promoter||Enzyme||Buffer||MilliQ||total
 +
|-
 +
|2||0.2||2||15.8||20
 +
|}
 +
====Checking tatABCD====
 +
{| class="wikitable"
 +
!tatABCD||Hind3||Buffer||MilliQ||total
 +
|-
 +
|5||0.2||2||12.8||20
 +
|}
 +
====PCR====
 +
{| class="wikitable"  
 +
! ||buffer||dNTPs||MgSO4||Primer-f||Primer-r||ColE1(6.5ng/µL) / TMAO||MilliQ||KOD Plus Neo||total  
 +
|-  
 +
|Kil||2.5||2.5||1.5||0.75||0.75||0.5||16||0.5||25
 +
|-
 +
|TMAO||2.5||2.5||1.5||0.75||0.75||0.5||16||0.5||25
 +
|}  
 +
Predenature 94℃,2min<br>
 +
Denature 98℃,10sec<br>
 +
Annealing 60℃,30sec<br>
 +
Extension 68℃,3min<br>
 +
→30cycles<br><br>
====Electrophoresis====
====Electrophoresis====
-
[[File:Electrophoresis031101.JPG|400px|thumb|right]]
+
*evernoteに写真なし！至急求む
-
1. 1kb ladder<br>
+
1. 1kb ladder<br>  
-
2〜11. pspA (969bp)<br>
+
2. Kil (649bp)<br>  
-
12. 1kb ladder<br>
+
3. TMAO (2720bp)<br>  
-
<br>The results were shown as photograph in the right.<br>
+
4. TMAO (Quick Taq)<br>  
-
<br>It seemed that there were shorter sample than expected sample, so we did electrophoresis with pspA which was product of PCR and pspA which had already cut with EcoR1 and Spe1.<br><br><br><br><br><br>
+
5. tatABCD (Quick Taq)<br>  
-
[[File:Electrophoresis031102.JPG|400px|thumb|right]]<br>
+
6. tatABCD (Hind3)<br>  
-
1.1kb ladder<br>
+
7. 1kb ladder<br>
-
2. pspA (PCR product)<br>
+
-
3, pspA (Eco, Spe)<br>
+
-
4〜6, pspA (colony PCR)<br>
+
-
7,1kb ladder<br>
+
-
<br>The results were shown as photograph in the right.<br>
+
-
==March 12==
+
==March 1==  
-
====Transformation====
+
====PCR====
-
{| class="wikitable"
+
*TMAO<br>
-
!DT(1ng/μL)||DT(0.1ng/μL)||Kil||lacP-torA||MilliQ||competent cell||total
+
Template is gDNA and product of colony PCR gel extraction<br>
-
|-
+
{| class="wikitable"  
-
|1||0||0||0||0||20||21
+
! ||Buffer||gNTPs||MgSO4||Primer-f||Primer-r||KOD Plus Neo||Template gDNA||product of gel extraction||DW||total  
-
|-
+
|-  
-
|0||1||0||0||0||20||21
+
|1||2.5||2.5||1.5||0.75||0.75||0.5||0.5||0||16||25
-
|-
+
|-  
-
|0||0||5||0||0||50||51
+
|2||2.5||2.5||1.5||0.75||0.75||0.5||0||2||14.5||25
-
|-
+
|}
-
|0||0||0||5||0||50||51
+
94℃, 2min<br>
-
|-
+
98℃, 10sec<br>
-
|0||0||0||0||1||20||21
+
60℃, 30sec<br>
-
|}
+
68℃, 1.5min<br>
 +
→25cycles<br><br>
 +
*Kil
 +
{| class="wikitable"
 +
!Buffer||dNTPs||MgSO4||primer-f||primer-r||colE1(6.5ng/µL)||KOD Plus Neo||MilliQ||total
 +
|-  
 +
|2.5||2.5||1.5||0.75||0.75||0.5||16||0.5||25
 +
|}
 +
94℃, 2min<br>
 +
98℃, 10sec<br>
 +
61℃, 30sec<br>
 +
68℃, 1min<br>
 +
→20cycles<br><br>
 +
====Electrophoresis====
 +
[[File:Electrophoresis030101.JPG|250px|thumb|right]]
 +
[[File:Electrophoresis030102.JPG|250px|thumb|right]]
 +
1. 1kb ladder<br>
 +
2. TMAO1 (gDNA)<br>
 +
3. TMAO2 (product of gel extraction)<br>
 +
4. Kil<br><br>
 +
====PCR====
 +
{| class="wikitable"
 +
!Buffer||dNTPs||MgSO4||primer-f||primer-r||Product of Purification||KOD Plus Neo||MilliQ||total
 +
|-
 +
|5||5||3||1.5||1.5||1||32||1||50
 +
|}  
 +
94℃, 2min<br>
 +
98℃, 10sec<br>
 +
61℃, 30sec<br>
 +
68℃, 30sec<br>
 +
20cycles<br>
 +
→Purification 230ng/µL<br><br>
====Restriction====
====Restriction====
-
{| class="wikitable"
+
{| class="wikitable"  
-
!pspA||EcoR1||Spe1||BufferM||BSA||MilliQ||total
+
!Kil||EcoR1||Spe1||BufferH||MilliQ||total  
-
|-
+
|-  
-
|10||0.5||0.5||3||0.3||15.7||30
+
|5||0.2||0.2||2||12.6||20
-
|}
+
|}  
-
at 37℃ for 4 hours<br>
+
incubate at 37℃, for 1.5 hours<br><br>  
-
→ We did purification and got 48.3ng/μL pspA.<br><br>
+
====PCR Purification====
====Ligation====
====Ligation====
-
{| class="wikitable"
+
{| class="wikitable"  
-
! |pspA||pSB1C3||MilliQ||Ligation High Ver.2||total
+
!Kil||pSB1C3||Ligation High||total  
-
|-
+
|-  
-
|1|4||2||0||3||9
+
|5||1||3||9  
-
|-
+
|}
-
|2|2||2||0||2||6
+
at 4℃, for overnight
-
|-
+
-
|3|0||2||2||2||6
+
-
|}
+
-
==March 13==
+
==March 2==  
-
====Miniprep====
+
====Ligation====
-
pSB1C3 74.2µg/mL  1.65 (260/280) 1.31 (260/230)
+
{| class="wikitable"
-
==March 14==
+
!Kil||pSB1C3||Ligation High Ver.2||total
 +
|-
 +
|5||1||3||9
 +
|}
 +
{| class="wikitable"
 +
!tatABCD||pSB1C3||Ligation||total
 +
|-
 +
|10||1||5||16
 +
|}
 +
at 16℃ for 1 hour<br><br>
 +
====Transformation====
 +
{| class="wikitable"
 +
!Kil||Kil(3/1,Ligation)||tatABCD||competet cell||total
 +
|-
 +
|1||0||0||10||11
 +
|-
 +
|0||1||0||10||11
 +
|-
 +
|0||0||1||10||11
 +
|}
 +
====PCR====
 +
{| class="wikitable"
 +
!Quick Taq||primer-r||primer-f||template||MilliQ||total
 +
|-
 +
|25||1||1||0.5||22.5||50
 +
|}
 +
====Electrophoresis====
 +
[[File:Electrophoresis030227.JPG|250px|thumb|right]]
 +
<br><br>
====Restriction====
====Restriction====
-
{| class="wikitable"
+
{| class="wikitable"
-
!pSB1C3||EcoR1||Spe1||BufferM||BSA||MilliQ||total
+
!pSB3C5-5||EcoR1||Pst1||BufferH||BSA||MilliQ||total
-
|-
+
|-
-
|20||0.2||0.2||4||0.4||15.2||40
+
|20||0.5||0.5||3||0.5||5.5||30
 +
|}
 +
at 37℃, for 2 hour<br><br>
 +
====Electrophoresis====
 +
[[File:Electrophoresis030225.JPG|250px|thumb|right]]
 +
1. 1kb ladder 2µL<br>
 +
2. pSB3C5 5µL + 6×Loading Buffer 1µL<br>
 +
・product of gel extraction(about 2700bp)<br>
 +
－30.9µg/mL<br><br>
 +
====Restriction====
 +
GFP1 ,2 ,3
 +
{| class="wikitable"
 +
!GFP||EcoR1||Pst1||Buffer||BSA||MilliQ||total
 +
|-
 +
|10||0.5||0.5||3||0.5||15.5||30
 +
|}
 +
at 37℃, for 2.5 hours<br><br>
 +
====Restriction====
 +
{| class="wikitable"
 +
!DT||EcoR1||Xba1||BufferM||BSA||MilliQ||total
 +
|-
 +
|10||0.2||0.2||3||0.3||16.3||30
 +
|}
 +
{| class="wikitable"
 +
!Constitutive Promoter||Spe1||Pst1||BufferM||BSA||MilliQ||total
 +
|-
 +
|15||0.2||0.2||3||0.3||11.3||30
 +
|}
 +
at 37℃, 2 hours<br>
 +
*J23117-1:135ng/µL, J23107-1:115ng/µL<br>
 +
*DT3→PCR Purification<br>
 +
*Promoter→Gel Extraction<br><br>
 +
====Checking TMAO====
 +
{| class="wikitable"
 +
!something seems to be TMAO||Buffer2||EcoR1||MilliQ||total
 +
|10||2||0.5||7.5||20
 +
|}
 +
at 37℃, for 1 hour<br><br>
 +
====Electrophoresis====
 +
[[File:Electrophoresis030226.JPG|250px|thumb|right]]
 +
1. 1kb ladder<br>
 +
2. GFP1 that had been cut by restriction enzyme<br>
 +
3. GFP2 that had been cut by restriction enzyme<br>
 +
4. GFP3 that had been cut by restriction enzyme<br>
 +
5. GFP1<br>
 +
6. GFP2<br>
 +
7. GFP3<br>
 +
8. TMAO (control)<br>
 +
9. TMAO (EcoR1)<br>
 +
10. DT (control)<br>
 +
11. DT (EcoR1, Xba1)<br>
 +
12. 1kb ladder<br><br>
 +
'''Checking tatABCD'''<br>
 +
{| class="wikitable"
 +
!Quick Taq||primer-f||primer-r||MilliQ||total
 +
|-
 +
|25||1||1|||23||50
 +
|}
 +
====Electrophoresis====
 +
[[File:Electrophoresis030228.JPG|250px|thumb|right]]
 +
<br><br><br><br><br><br><br><br><br><br><br><br>
 +
 
 +
==March 3==
 +
====PCR====
 +
{| class="wikitable" style="text-align: right"
 +
! ||template||buffer||dNTPs||MgSO4||VF||VR||KOD plus||MilliQ||total
 +
 
 +
|-
 +
|1||1||5||5||3||1.5||1.5||1||32||50
 +
|-
 +
|2||2||5||5||3||1.5||1.5||1||31||50
 +
|}
 +
94℃, 2min<br>
 +
98℃, 10sec<br>
 +
50℃, 30sec<br>
 +
68℃, 1min<br>
 +
→30cycles<br><br>
 +
====Miniprep====
 +
==March 4==
 +
====Sequence of tatABCD====
 +
{| class="wikitable" style="text-align: right;"
 +
!Quick Taq||primer-f||promer-r sequence||template||MilliQ||total
 +
|-
 +
|25||1||1||1||23||50
 +
|}
 +
{| class="wikitable" style="text-align: right;"
 +
!Quick Taq||primer-f sequence||primer-r||template||MilliQ||total
 +
|-
 +
|25||1||1||1||23||50
 +
|}
 +
====Colony PCR of TMAO====
 +
{| class="wikitable" style="text-align: right;"
 +
!buffer||dNTPs||NgSO4||primer-f||primerr-r||KOD plus||MilliQ||total
 +
|-
 +
|5||5||4||1.5||1.5||1||32||50
 +
|}
 +
→ethanol precipitation<br>
 +
94℃, 2min<br>
 +
94℃, 30sec<br>
 +
55℃, 30sec<br>
 +
68℃, 2.5min<br>
 +
→25cycles<br><br>
 +
====Electrophoresis====
 +
1. 1kb ladder<br>
 +
2. tatABCD1<br>
 +
3. tatABCD2<br>
 +
4. TMAO<br>
 +
5. 1kb ladder<br><br>
 +
====Restriction====
 +
{| class="wikitable"
 +
!TMAO||EcoR1||BufferH||BSA||MilliQ||total
 +
|-
 +
|10||0.2||3||0.3||16.5||30
 +
|}
 +
{|class="wikitable"
 +
!TMAO||Xba1||Pst1||BudderM||BSA||MilliQ||total
 +
|-
 +
|10||0.2||0.2||3||0.3||16.3||30
 +
|}
 +
at 37℃ for 1 hour<br><br>
 +
====Electrophoresis====
 +
<br>
 +
====Transformation====
 +
{|class="wikitable"
 +
!pSB1C3||competent cell(made at 2/8)||total
 +
|-
 +
|5||100||105
 +
|}
 +
==March 5==
 +
====Restriction====
 +
{| class="wikitable"
 +
!pSB1C3(Xba1, Spe1)||Pst1||BufferH||BSA||MilliQ||total
 +
|-
 +
|10||0.2||2||0.2||7.6||20
 +
|-
 +
!pSB1C3(Xba1, Spe1)||EcoR1||BufferH||BSA||MilliQ||total
 +
|-
 +
|10||0.2||2||0.2||7.6||20
 +
|}
 +
at 37℃ for 1 hour<br>
 +
→Then we did ethanol precipitation<br><br>
 +
====Ligation====
 +
{| class="wikitable"
 +
!Kil(EcoR1, Spe1)||pSB1C3(EcoR1)||Ligation High||total
 +
|-
 +
|5||1||3||9
 +
|}
 +
at 16℃ for 1 hour<br><br>
 +
====Transformation====
 +
{| class="wikitable"
 +
!Kil||competent cell||total
 +
|-
 +
|1||10||11
 +
|}
 +
We used commercially available competent cells in this time.<br><br>
 +
====PCR====
 +
TMAO<br>
 +
{| class="wikitable"
 +
!buffer||dNTPs||MgSO4||Primer-f||Primer-r||Template||KOD plus||MilliQ||total
 +
|-
 +
|5||5||3||1.5||1.5||1||1||32||50
 +
|}
 +
====Electrophoresis====
 +
(31)<br><br>
 +
====Restriction====
 +
{| class="wikitable"
 +
! ||LacP||pSB3C5||EcoR1||Pst1||BufferH||BSA||MilliQ||total
 +
|-
 +
|1||20||0||0.5||0.5||3||0.5||5.5||30
 +
|-
 +
|2||0||20||0.5||0.5||3||0.5||5.5||30
 +
|}
 +
at 37℃  for 1 hour<br>
 +
1→Ethanol precipitation　45.4µg/mL<br>
 +
2→Gel extraction　38.7µg/mL<br><br>
 +
====Ligation====
 +
{| class="wikitable"
 +
!LacP||pSB3C5||Ligation High||total
 +
|-
 +
|10||2||6||18
 +
|}
 +
at 4℃ for overnight<br><br>
 +
====Transformation====
 +
{| class="wikitable"
 +
!LacP+pSB3C5||competent cell||total
 +
|-
 +
|1||10||11
 +
|}
 +
on ice for 30 mins.<br>
 +
heat shock at 42℃ for 60secs<br>
 +
on ice for 2 mins.<br>
 +
After we incubate with 200µL of SOC culture for 1 hour, we did plating on LB culture with CP<br><br>
 +
====Restriction====
 +
{| class="wikitable"
 +
!GFP Plasmid||EcoR1||Spe1||Buffer2||BSA||MilliQ||total
 +
|-
 +
|10||0.5||0.5||3||0.5||15.5||30
 +
|}
 +
at 37℃ for 2 hours
 +
====Electrophoresis====
 +
1. Ladder 2µL<br>
 +
2. GFP Plasmid (already restricted) 2µL + Loading Dye 2µL + MilliQ 8µL<br>
 +
3. Ladder 2µL<br>
 +
====PCR====
 +
torA signal and pspA
 +
pspAはコロニーPCR<br>
 +
{| class="wikitable"
 +
!Buffer||dNTPs||MgSO4||Primer-f||Primer-r||template(TMAO)||KOD plus||MilliQ||total
 +
|-
 +
|2.5||2.5||1.5||0.75||0.75||0.5||0.5||16||25
 +
|}
 +
94℃, 2min<br>
 +
98℃, 10sec<br>
 +
60℃, 30sec<br>
 +
68℃, 30sec<br>
 +
====electrophoresis====
 +
1. 100bp Ladder<br>
 +
2. torA signal (272bp)<br>
 +
3. pspA (969bp)<br>
 +
4. 100bp Ladder<br>
 +
==March 6==
 +
====Restriction====
 +
{| class="wikitable"
 +
!GFP||EcoR1||Spe1||BufferM||BSA||MilliQ||total
 +
|-
 +
|12||0.5||0.5||3||0.5||13.5||30
 +
|-
 +
|}
 +
We did incubate at 37℃ for 1.5hours.<br>
 +
And we did gel extraction on 3/7 and get 40.0μg/mL GFP.<br><br>
 +
====PCR====
 +
{| class="wikitable"
 +
!buffer||dNTPs||MgSO4||Primer-f||Primer-r||template||KOD plus neo||MilliQ||total
 +
|-
 +
|5||5||3||1.5||1.5||0.5||1||32.5||50
 +
|}
 +
94℃　2min<br>
 +
98℃　10sec<br>
 +
60℃　30sec<br>
 +
68℃　(torA 10sec / pspA 30sec)    25 cycles<br>
 +
We used product of PCR on 3/5 of torA and pspA as template.<br><br>
 +
====Restriction====
 +
{| class="wikitable"
 +
!Kil||EcoR1||Spe1||BufferM||BSA||MilliQ||total
 +
|-
 +
|10||0.3||0.3||3||0.3||16.1||30
 +
|}
 +
{| class="wikitable"
 +
!DT||EcoR1||Xba1||Buffer2||BSA||MilliQ||total
 +
|-
 +
|10||0.5||0.5||3||0.5||15.5||30
 +
|}
 +
at 37℃ for 2 hours<br>
 +
→ purification 37.7ng/μL<br><br>
 +
====Ligation====
 +
{| class="wikitable"
 +
!Kil||pSB1C3||Ligation High Ver.2||total
 +
|-
 +
|4||2||3||9
 +
|}
 +
*Kil : 350fmol<br>
 +
*pSB1C3 : 29fmol<br>
 +
at 16℃ for overnight<br>
 +
==March 7==
 +
====Electrophoresis====
 +
[[File:Electrophoresis0307.JPG|400px|thumb|right]]<br>
 +
1. 1kb ladder 2µL<br>
 +
2. pspA (PCR product)2.5µL + Loading Dye 0.5µL<br>
 +
3. GFP 30µL + Loading Dye 6µL<br>
 +
4. 1kb ladder 2µL<br>
 +
*The GFP was gel extracted on 3/6 and concentrated by Vacuum in 150µg/mL<br><br>
 +
====Ligation====
 +
{| class="wikitable"
 +
!VectorDNA||GFP||Ligation High Ver.2||total
 +
|-
 +
|5||15||10||30
 +
|}
 +
====Restriction====
 +
{| class="wikitable"
 +
!torA||EcoR1||Spe1||bufferM||BSA||MilliQ||total
 +
|-
 +
|10||0.3||0.3||3||0.3||16.1||30
 +
|}
 +
{| class="wikitable"
 +
!pspA||EcoR1||Spe1||bufferM||BSA||MilliQ||total
 +
|-
 +
|5||0.3||0.3||3||0.3||21.1||30
 +
|}
 +
at 37℃ for 1.5 hours<br><br>
 +
====Purification====
 +
torA→31.8ng/µL<br>
 +
pspA→49.3ng/µL<br><br>
 +
====Ligation====
 +
{| class="wikitable"
 +
!torA||pSB1C3||Ligation High Ver.2||total
 +
|-
 +
|3||3||3||9
 +
|}
 +
{| class="wikitable"
 +
!pspA||pSB1C3||Ligation High Ver.2||total
 +
|-
 +
|4||2||3||9
 +
|}
 +
at 4℃, for overnight<br>
 +
*torA→31.8ng/µL×3µL=95.4ng=0.529pmol<br>
 +
*pSB1C3→19.4ng/µL×3µL=58.2ng=0.042pmol<br>
 +
*pspA→49.3ng/µL×4µL=197.2ng=0.308pmol<br>
 +
*pSB1C3→19.4ng/µL×2µL=38.8ng=0.029pmol<br>
 +
==March 8==
 +
====Restriction====
 +
{| class="wikitable"
 +
!pSB4K5||EcoR1||Spe1||BufferM||BSA||MilliQ||total
 +
|-
 +
|20||0.2||0.2||3||0.2||6.4||30
 +
|-
 +
|}
 +
at 37℃ for 1 hour.<br>
 +
→Purification : 36.6ng/µL<br><br>
 +
====Ligation====
 +
{| class="wikitable"
 +
!Kil||pSB4K5||Ligation High Ver.2||total
 +
|-
 +
|10||1||5||16
 +
|}
 +
at 4℃ for overnight<br>
 +
*Kil→37.7ng/µL×10µL=377ng=879fmol<br>
 +
*pSB4K5→36.6ng/µL×1µL=36.6ng=86fmol<br><br>
 +
====Liquid culture====
 +
LacP + pSB3C5 -1, 2<br><br>
 +
====Transformation====
 +
{| class="wikitable"
 +
!torA||pspA||competent cell||total
 +
|-
 +
|1||0||10||11
 +
|-
 +
|0||1||10||11
 +
|}
 +
We use commercially available competent cells in this time.<br>
 +
==March 9==
 +
====Restriction====
 +
{| class="wikitable"
 +
!tatABCD||Xba1||Pst1||BufferM||BSA||MilliQ||total
 +
|10||0.2||0.2||3||0.3||16.3||30
 +
|}
 +
at 37℃ for 1hour<br>
 +
→purification : 75.0μg/mL    (1.11  260/280 , 0.81  260/230)<br><br>
 +
====Miniprep====
 +
LacP + pSB3C5 1    80.5μg/mL    (1.78  260/280 , 2.00  260/230)<br>
 +
LacP + pSB3C5 2    107.2μg/mL  (1.83  260/280 , 1.90  260/230)<br><br>
 +
====Colony PCR====
 +
{| class="wikitable"
 +
!Quick Taq||VF||VR||MilliQ||total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
94℃ 2min<br>
 +
94℃ 30sec<br>
 +
55℃ 30sec<br>
 +
68℃ 6sec<br>
 +
25cycles<br><br>
 +
====Ligation====
 +
{| class="wikitable"
 +
!tatABCD||constP J23107||Ligation High Ver.2||total
 +
|-
 +
|5||1||3||9
 +
|}
 +
tatABCD : 227fmol<br>
 +
constP J23107 : 21fmol<br><br>
 +
====Transformation====
 +
{| class="wikitable"
 +
! ||pspA||torA||Kil||competent cell
 +
|-
 +
|1||1||0||0||10
 +
|-
 +
|2||0||1||0||10
 +
|-
 +
|3||0||0||1||10
 +
|}
 +
====Miniprep====
 +
4mL of plusgrow which had been cultured for overnight.<br>
 +
pSB4K5 : 80.5μg/mL<br>
 +
==March 10==
 +
====Screening PCR====
 +
{| class="wikitable"
 +
!Quick Taq||VF||VR||MilliQ||total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
[[File:Electrophoresis0310.JPG|400px|thumb|right]]
 +
Then we did electrophoresis to confirm.<br>
 +
1.1kb ladder<br>
 +
2.kil (649bp)<br>
 +
3,4,5, pspA (969bp)<br>
 +
6,1kb ladder<br><br>
 +
1, 100bp ladder<br>
 +
2,3,4, torA signal<br><br>
 +
====Restriction====
 +
{| class="wikitable"
 +
!LacP-pSB3C5||Spe1||Pst1||BufferM||BSA||MilliQ||total
 +
|-
 +
|10||0.2||0.2||2||0.2||7.4||20
 +
|}
 +
for 2.5 hours at 37℃<br>
 +
→purification  29.0ng/μL<br>
 +
{| class="wikitable"
 +
!torA||Xba1||Pst1||BufferM||BSA||MilliQ||total
 +
|-
 +
|10||0.2||0.2||2||0.2||7.4||20
 +
|}
 +
for 2.5 hours at 37℃<br>
 +
→purification  91.8ng/μL<br><br>
 +
====Ligation====
 +
{| class="wikitable"
 +
!torA||LacP-pSB3C5||Ligation High Ver.2||total
 +
|-
 +
|4||2||3||9
 +
|}
 +
torA : 929fmol<br>
 +
LacP-pSB3C5 : 284fmol<br>
 +
for overnight at 4℃
 +
==March 11==
 +
====Miniprep====
 +
We used 3μL of plus grow that we had cultured for overnight.<br>
 +
torA : 62.8ng/μL<br><br>
 +
====Screening PCR====
 +
{| class="wikitable"
 +
!Quick Taq||VF||VR||MilliQ||total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
====Electrophoresis====
 +
[[File:Electrophoresis031101.JPG|400px|thumb|right]]
 +
1. 1kb ladder<br>
 +
2〜11. pspA (969bp)<br>
 +
12. 1kb ladder<br>
 +
<br>The results were shown as photograph in the right.<br>
 +
<br>It seemed that there were shorter sample than expected sample, so we did electrophoresis with pspA which was product of PCR and pspA which had already cut with EcoR1 and Spe1.<br><br><br><br><br><br>
 +
[[File:Electrophoresis031102.JPG|400px|thumb|right]]<br>
 +
1.1kb ladder<br>
 +
2. pspA (PCR product)<br>
 +
3, pspA (Eco, Spe)<br>
 +
4〜6, pspA (colony PCR)<br>
 +
7,1kb ladder<br>
 +
<br>The results were shown as photograph in the right.<br>
 +
==March 12==
 +
====Transformation====
 +
{| class="wikitable"
 +
!DT(1ng/μL)||DT(0.1ng/μL)||Kil||LacP-torA||MilliQ||competent cell||total
 +
|-
 +
|1||0||0||0||0||20||21
 +
|-
 +
|0||1||0||0||0||20||21
 +
|-
 +
|0||0||5||0||0||50||51
 +
|-
 +
|0||0||0||5||0||50||51
 +
|-
 +
|0||0||0||0||1||20||21
 +
|}
 +
====Restriction====
 +
{| class="wikitable"
 +
!pspA||EcoR1||Spe1||BufferM||BSA||MilliQ||total
 +
|-
 +
|10||0.5||0.5||3||0.3||15.7||30
 +
|}
 +
at 37℃ for 4 hours<br>
 +
→ We did purification and got 48.3ng/μL pspA.<br><br>
 +
====Ligation====
 +
{| class="wikitable"
 +
! |pspA||pSB1C3||MilliQ||Ligation High Ver.2||total
 +
|-
 +
|1|4||2||0||3||9
 +
|-
 +
|2|2||2||0||2||6
 +
|-
 +
|3|0||2||2||2||6
 +
|}
 +
==March 13==
 +
====Miniprep====
 +
pSB1C3  74.2µg/mL  1.65 (260/280)  1.31 (260/230)
 +
==March 14==
 +
====Restriction====
 +
{| class="wikitable"  
 +
!pSB1C3||EcoR1||Spe1||BufferM||BSA||MilliQ||total  
 +
|-  
 +
|20||0.2||0.2||4||0.4||15.2||40  
 +
|}
 +
We did gel extraction and got 47.2ng/µL pSB1C3 but we did not cut out RFP.<br><br>
 +
====Ligation====
 +
{| class="wikitable"
 +
!torA||pSB1C3||Ligation High Ver.2||total
 +
|-
 +
|4||2||3||9
 +
|-
 +
!MilliQ||pSB1C3||Ligation High Ver.2||total
 +
|-
 +
|4||2||3||9
 +
|}
 +
at 16℃, for 1 hour<br>
 +
*torA : 0.707pmol<br>
 +
*pSB1C3 : 0.068pmol<br><br>
 +
====Liquid culture====
 +
We cultured LacP-pSB3C5 1,2,3,4,5 (CP tolerance)on culture with Amp.<br>
 +
→Only 4 which did not be cultured succeeded.
 +
==March 15==
 +
====Liquid culture====
 +
We cultured LacP-pSB3C5 6,7,8,9,10 on culture with ampicillin.<br>
 +
→6,8,9,10 were succeeded.<br><br>
 +
====Restriction====
 +
{| class="wikitable"
 +
!GFP||EcoR1||Spe1||Buffer2||BSA||MilliQ||total
 +
|-
 +
|10||0.5||0.5||3||0.5||15.5||30
 +
|}
 +
{| class="wikitable"
 +
!DT||EcoR1||Xba1||Buffer2||BSA||MilliQ||total
 +
|-
 +
|10||0.5||0.5||3||0.5||15.5||30
 +
|}
 +
for 2 hours at 37℃.<br><br>
 +
====Miniprep====
 +
pspA (pSB1C3)  40.5ng/µL<br><br>
 +
====Ligation====
 +
{| class="wikitable"
 +
!pspA||DT||Ligation High Ver.2||total
 +
|-
 +
|5||1.5||3||9.5
 +
|}
 +
*pspA : 385fmol<br>
 +
*DT : 36fmol<br><br>
 +
====Transformation====
 +
{| class="wikitable"
 +
!J23107-tatABCD||DT (0.1ng/µL)||DT (0.01ng/µL)||pspA-DT||competent cells on 3/15||total
 +
|-
 +
|2||0||0||0||20||22
 +
|-
 +
|0||2||0||0||20||22
 +
|-
 +
|0||0||2||0||20||22
 +
|-
 +
|0||0||0||2||20||22
 +
|}
 +
====Screening PCR====
 +
Kil, pspA and torA<br>
 +
{| class="wikitable"
 +
!Quick Taq||Primer-r||Primer-f||MilliQ||total
 +
|25||1||1||23||50
 +
|}
 +
==March 16==
 +
====Miniprep====
 +
torA (pSB1C3)  68.8ng/µL<br>
 +
Kil (pSB4K5)  92.7ng/µL<br>
 +
torA was red for some reason. We do not know why.<br><br>
 +
====Colony PCR====
 +
{| class="wikitable"
 +
!Quick Taq||Primer-r||Primer-f||MilliQ||total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
94℃, 2min<br>
 +
94℃, 30sec<br>
 +
55℃, 30sec<br>
 +
68℃, 6sec<br>
 +
→25cycles<br><br>
 +
====Electrophoresis====
 +
[[File:Electrophoresis0316.JPG|400px|thumb|right]]
 +
The results were shown as photograph in the right.<br><br>
 +
====Checking Transformation Efficiency====
 +
competent cells that were made on March 15.<br>
 +
DNA : 0.02ng → 668 colonies  Transformation Efficiency : 3.3×10^7<br>
 +
DNA : 0.2ng → 1739 colonies  Transformation Efficiency : 8.7×10^6<br><br>
 +
====Restriction====
 +
{| class="wikitable"
 +
!pSB1C3||EcoR1||Spe1||BSA||BufferM||BufferH||MilliQ||total
 +
|-
 +
|20||0.2||0.2||0.3||3||0||6.3||30
 +
|-
 +
|5||0.2||0||0.2||0||2||12.6||20
 +
|}
 +
at 37℃, for 2 hours.<br>
 +
We did gel extraction for product with EcoR1, Spe1. We got 46.1ng/µL pSB1C3.<br>
 +
{| class="wikitable"
 +
!Kil(pSB4K5)||EcoR1||Pst1||BSA||BufferH||MilliQ||total
 +
|-
 +
|5||0.2||0.2||0.2||2||12.4||20
 +
|-
 +
|5||0.2||0||0.2||2||12.6||20
 +
|}
 +
for overnight at 37℃.<br>
 +
We did this to confirm.<br>
 +
{| class="wikitable"
 +
!pspA (pSB1C3)||EcoR1||Pst1||BSA||BufferH||MilliQ||total
 +
|-
 +
|5||0.2||0.2||0.2||2||12.4||20
 +
|-
 +
|5||0.2||0||0.2||2||12.6||20
 +
|}
 +
for overnight at 37℃.<br>
 +
We did this to confirm.<br><br>
 +
====Ligation====
 +
{| class="wikitable"
 +
!torA||pSB1C3||Ligation High Ver.2||total
 +
|-
 +
|4||2||3||9
 +
|}
 +
{| class="wikitable"
 +
!MilliQ||pSB1C3||Ligation High Ver.2||total
 +
|-
 +
|4||2||3||9
 +
|}
 +
at 16℃, for overnight<br>
 +
*torA→767fmol<br>
 +
*pSB1C3→68fmol<br>
 +
==March 17==
 +
====Miniprep====
 +
J23107-tatABCD  72.7ng/µL<br>
 +
pspA-DT  50.5ng/µL<br><br>
 +
====Checking the Insert====
 +
{| class="wikitable"
 +
!J21037-tatABCD||EcoR1||Pst1||BSA||BufferH||MilliQ||total
 +
|-
 +
|5||0.2||0.2||0.2||2||12.4||20
 +
|-
 +
|5||0.2||0||0.2||2||12.6||20
 +
|}
 +
Success.
 +
{| class="wikitable"
 +
!pspA-DT||EcoR1||Pst1||BSA||BufferH||MilliQ||total
 +
|-
 +
|5||0.2||0.2||0.2||2||12.4||20
 +
|-
 +
|5||0.2||0||0.2||2||12.6||20
 +
|}
 +
Failed.
 +
==March 19==
 +
====Restriction====
 +
{| class="wikitable"
 +
!DT||EcoR1||Xba1||BSA||BufferM||MilliQ||total
 +
|-
 +
|10||0.2||0.2||0.3||3||16.3||30
 +
|}
 +
We did Gel extraction and got 17.2ng/µL of DT.<br><br>
 +
====Ligation====
 +
{| class="wikitable"
 +
!Kil||DT||Ligation High Ver.2||total
 +
|-
 +
|10||2||6||18
 +
|}
 +
We did this for an hour at 16℃.<br><br>
 +
====Restriction====
 +
{| class="wikitable"
 +
!GFP||EcoR1||Spe1||BSA||BufferM||MilliQ||total
 +
|-
 +
|10||0.5||0.5||0.5||3||15.5||30
 +
|}
 +
We did this for 4 hours at 37℃<br><br>
 +
====Ligation====
 +
{| class="wikitable"
 +
!pspA||DT||Ligation High Ver.2||total
 +
|-
 +
|5||5||5||15
 +
|}
 +
{| class="wikitable"
 +
!pspA||pSB1C3||Ligation High Ver.2||total
 +
|-
 +
|4||1||3||8
 +
|}
 +
We did these for an hour at 16℃.<br>
 +
*pspA (5µL)→377fmol<br>
 +
*DT→39fmol<br>
 +
*pspA (4µL)→339fmol<br>
 +
*pSB1C3→34fmol<br><br>
 +
====Transformation====
 +
pspA-DT, pspA (pSB1C3), torA (pSB1C3) and GFP-DT
 +
==March 20==
 +
====Screaning PCR====
 +
{| class="wikitable"
 +
!Quick Taq||Primer-R||Primer-F||MilliQ||total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
*pspA→○<br>
 +
*pspA-DT→○<br>
 +
*GFP-DT→○<br>
 +
*torA→×<br>
 +
*Kil-DT 6 of 8 sumples→○<br>
 +
{| class="wikitable"
 +
!Quick Taq||VR||VF||MilliQ||total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
*pspA→○<br>
 +
*pspA-DT→×<br><br>
 +
====Restriction====
 +
{| class="wikitable"
 +
!torA||EcoR1||Spe1||BSA||BufferM||MilliQ||total
 +
|-
 +
|10||0.3||0.3||0.3||3||16.1||30
 +
|}
 +
{| class="wikitable"
 +
!DT||EcoR1||Xba1||BSA||BufferM||MilliQ||total
 +
|-
 +
|10||0.2||0.2||0.3||3||16.3||30
 +
|}
 +
We did this for overnight at 37℃. And we did purification.<br>
 +
torA  34.2ng/µL<br>
 +
DT  28.0ng/µL<br><br>
 +
==March 21==
 +
====Miniprep====
 +
GFP-DT-1 : 77.7ng/µL<br>
 +
GFP-DT-2 : 67.6ng/µL<br><br>
 +
====Restriction====
 +
{| class="wikitable"
 +
!pSB1C3||EcoR1||Spe1||BSA||BufferM||MilliQ||total
 +
|-
 +
|10||0.2||0.2||0.3||3||16.3||30
 +
|-
 +
|5||0.2||0||0.2||2||12.6||20
 +
|}
 +
We did this for 3 hours at 37℃, and then we did gel extraction. We got 25.1ng/µL pSB1C3<br><br>
 +
{| class="wikitable"
 +
!GFP-DT||EcoR1||Pst1||Xba1||BSA||BufferM||MilliQ||total
 +
|-
 +
|5||0.2||0.2||0||0.2||2||12.4||20
 +
|-
 +
|5||0.2||0||0||0.2||2||12.6||20
 +
|-
 +
|10||0.2||0||0.2||0.3||3||16.3||30
 +
|}
 +
We did this for 3 hours at 37℃, and then we did Purification. We got 30.7ng/µL GFP-DT.<br><br>
 +
====Ligation====
 +
{| class="wikitable"
 +
! ||torA||pSB1C3||pspA||DT||GFP-DT||Ligation High Ver.2||total
 +
|-
 +
|1||4||1||0||0||0||3||8
 +
|-
 +
|2||0||1||7||0||0||4||12
 +
|-
 +
|3||0||0||5||3||0||4||12
 +
|-
 +
|4||3||0||0||0||5||4||12
 +
|}
 +
We did this for an hour at16℃.
 +
==March 22==
 +
====PCR====
 +
We did PCR to amplify torA_signal that was product of PCR at March 5 with redesigned primers.<br>
 +
{| class="wikitable"
 +
!Buffer||dNTPs||MgSO4||Primer-F||Primer-R||Template||MilliQ||KOD plus neo||total
 +
|-
 +
|5||5||3||1.5||1.5||0.5||32.5||1||50
 +
|}
 +
94℃, 2min<br>
 +
98℃, 10sec<br>
 +
60℃, 30sec<br>
 +
68℃, 10sec<br>
 +
→30cycles<br>
 +
→Purification  110.7ng/µL<br><br>
 +
====Restriction====
 +
{| class="wikitable"
 +
!torA||EcoR1||Spe1||BSA||BufferM||MilliQ||total
 +
|-
 +
|10||0.2||0.2||0.3||3||16.3||30
 +
|}
 +
====Ligation====
 +
{| class="wikitable"
 +
! ||torA||pSSB1C3||pspA||DT||GFP-DT||Ligation high||total
 +
|-
 +
|1||4||3||0||0||0||4||11
 +
|-
 +
|2||3||0||0||0||3||3||9
 +
|-
 +
|3||0||0||5||5||0||5||15
 +
|}
 +
*torA (4µL)→512fmol<br>
 +
*pSB1C3→54fmol<br>
 +
*torA (3µL)→384fmol<br>
 +
*GFP-DT→36fmol<br>
 +
*pspA→377fmol<br>
 +
*DT→65fmol<br><br>
 +
 
 +
==March 23==
 +
====Screening PCR====
 +
torA (pSB1C3), torA-GFP-DT and pspA-DT<br>
 +
{| class="wikitable"
 +
!Quick Taq||VF||VR||MilliQ||total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
E.coli in liquid culture that had pspA(pSB1C3) also expressed RFP.<br><br>
 +
====Restriction====
 +
{| class="wikitable"
 +
!pspA||EcoR1||Spe1||BufferM||BSA||MilliQ||total
 +
|-
 +
|5||0.3||0.3||3||0.3||21.1||30
 +
|}
 +
And then we did ethanol precipitation<br><br>
 +
====Ethanol precipitation====
 +
pspA 11.5ng/µL.<br><br>
 +
====Miniprep====
 +
LacP+pSB3C5-8  77.9µg/mL<br>
 +
LacP+pSB3C5-10  69.0µg/mL<br>
 +
Kil+DT-4  58.0µg/mL<br>
 +
Kil+DT-7  15.2µg/mL<br><br>
 +
====Restriction====
 +
{| class="wikitable"
 +
!LacP+pSB3C5-8||Spe1||Pst1||Buffer2||BSA||MilliQ||total
 +
|-
 +
|20||0.5||0.5||3||0.5||5.5||30
 +
|}
 +
{| class="wikitable"
 +
!Kil+DT-4||Xba1||Pst1||Buffer2||BSA||MilliQ||total
 +
|-
 +
|20||0.5||0.5||3||0.5||5.5||30
 +
|}
 +
at 37℃ for 1.5 hours<br>
 +
And then we did Gel extraction.<br><br>
 +
====Gel Extraction====
 +
LacP+pSB3C5-8  40.4µg/mL<br>
 +
Kil+DT-4  26.8µg/mL<br>
 +
 
 +
==March 26==
 +
====Miniprep====
 +
torA(pSB1C3) 18.5[ng/µL]<br>
 +
torA-GFP-DT 20.0[ng/µL]<br><br>
 +
====Restriction====
 +
{| class="wikitable"
 +
!torA(pSB1C3)||EcoR1||Pst1||BufferH||BSA||MilliQ||total
 +
|-
 +
|5||0.2||0.2||2||0.2||12.4||20
 +
|-
 +
|5||0.2||0||2||0.2||12.6||20
 +
|}
 +
{| class="wikitable"
 +
!torA-GFP-DT||EcoR1||Xba1||Pst1||BufferH||BufferM||BSA||MilliQ||total
 +
|-
 +
|5||0.2||0||0.2||2||0||0.2||12.4||20
 +
|-
 +
|5||0.2||0||0||2||0||0.2||12.6||20
 +
|-
 +
|20||0||0.2||0.2||0||3||0.3||6.3||30
 +
|}
 +
We did Gel extraction and then got ??? 28.7[ng/µL]<br><br>
 +
====Ligation====
 +
{| class="wikitable"
 +
!LacP (pSB3C5)||torA-GFP-DT||Ligation High Ver.2||total
 +
|-
 +
|1||5||3||9
 +
|}
 +
*LacP : 22fmol<br>
 +
*torA-GFP-DT : 197fmol<br>
 +
{| class="wikitable"
 +
!pspA||pSB1C3||Ligation High Ver.2||total
 +
|-
 +
|10||1||5||16
 +
|}
 +
{| class="wikitable"
 +
!pspA||DT||Ligation High Ver.2||total
 +
|-
 +
|10||1||5||16
 +
|}
 +
*pspA : 180fmol<br>
 +
*pSB1C3 : 18fmol<br>
 +
*DT : 16fmol<br>
 +
{| class="wikitable"
 +
!LacP(pSB3C5)||Kil-DT||Ligation High Ver.2||total
 +
|-
 +
|1||5||3||9
 +
|}
 +
for 2 hours at 16℃<br><br>
 +
====Transformation====
 +
{| class="wikitable"
 +
!LacP-Kil-DT||competent cell||total
 +
|-
 +
|1||10||11
|}
|}
-
We did gel extraction and got 47.2ng/µL pSB1C3 but we did not cut out RFP.<br><br>
+
 
-
====Ligation====
+
==March 27==
-
{| class="wikitable"
+
====Miniprep====
-
!torA||pSB1C3||Ligation High Ver.2||total
+
We retryed miniprep of torA(pSB1C3).<br>
-
|-
+
We got torA and its concentration was 39.3[ng/µL].<br><br>  
-
|4||2||3||9
+
====Transformation====  
-
|-
+
{| class="wikitable"
-
!MilliQ||pSB1C3||Ligation High Ver.2||total
+
!Name||Well||Sample||Competent Cells||Total||Plate||Colony
-
|-
+
|-  
-
|4||2||3||9
+
|BBa_K117004 ||14J(2011 plate2)||5||20||?||?||?
 +
|}
 +
We added 100[µL] of culture medium before we started culturing the E.coli.<br><br>
 +
====Screening PCR====
 +
{| class="wikitable"
 +
!Quick Taq||VF2||VR||MilliQ||Total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
LacP-torA-GFP-DT 1, 3, 5, 6, 8, 9 was successful.<br>
 +
pspA-DT was failed.<br>
 +
E.coli that had pspA(pSB1C3) did not make any colony.<br>
 +
LacP-Kil-DT 1,2,3,4,5,6,7,8 was failed.<br><br>
 +
====Liquid culture====
 +
LacP-torA-GFP-DT<br>
 +
 
 +
==July 23==
 +
====Transformation====
 +
{|class="wikitable"
 +
!DT(64.4ng/μl)||Competent cell||Plating in SOC medium||Total
 +
|-  
 +
|1||20||100||121
|}
|}
-
at 16℃, for 1 hour<br>
 
-
*torA : 0.707pmol<br>
 
-
*pSB1C3 : 0.068pmol<br><br>
 
-
====Liquid culture====
 
-
We cultured Lacp-pSB3C5 1,2,3,4,5 (CP tolerance)on culture with Amp.<br>
 
-
→Only 4 which did not be cultured succeeded.
 
-
==March 15==
+
==July 24==  
-
====Liquid culture====
+
 
-
We cultured Lacp-pSB3C5 6,7,8,9,10 on culture with ampicillin.<br>
+
 
-
→6,8,9,10 were succeeded.<br><br>
+
 
-
====Restriction====
+
-
{| class="wikitable"
+
 
-
!GFP||EcoR1||Spe1||Buffer2||BSA||MilliQ||total
+
 
-
|-
+
 
-
|10||0.5||0.5||3||0.5||15.5||30
+
 
 +
 
 +
 
 +
====Plating in SOC medium====  
 +
 
 +
 
 +
 
 +
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
==July 25==
 +
====Transformation====
 +
*BBa_J23113 from iGEM Kit
 +
*LacP from iGEM Kit
 +
 
 +
==July 25==
 +
 
 +
 
 +
 
 +
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
==July 26==
 +
 
 +
 
 +
 
 +
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
==July 27==
 +
====Restriction Enzyme Processing====  
 +
{|class="wikitable"  
 +
!Kil +DT (44.0 ng/μl)||10xBuffer2||BSA||Xbal||MilliQ||Total
 +
|-  
 +
|13.6||3.0||0.5||0.5||11.9||30.0
 +
|}
 +
{|class="wikitable"
 +
!LacP(28.7ng/μl)||10xBuffer2 ||BSA||Spe1||Pst1||Total
 +
|-
 +
|20.9||3.0||0.5||0.5||4.6||30.0
|}
|}
-
{| class="wikitable"
+
 
-
!DT||EcoR1||Xba1||Buffer2||BSA||MilliQ||total
+
==July 30==
-
|-
+
====Ligation====
-
|10||0.5||0.5||3||0.5||15.5||30
+
{| class="wikitable"  
 +
!kil+DT(Xba1,Pst1)||LacP(Spe1,Pst1)||Ligation High ||Total
 +
|-  
 +
|30||6||36||72
|}
|}
-
for 2 hours at 37℃.<br><br>
+
 
-
====Miniprep====
+
==July 31==
-
pspA (pSB1C3) 40.5ng/µL<br><br>
+
====Restriction Enzyme Processing ====
-
====Ligation====
+
{| class="wikitable"
-
{| class="wikitable"
+
!torA-GFP+DT||10×M Buffer||BSA||MilliQ||Xba1||Pst1||Total
-
!pspA||DT||Ligation High Ver.2||total
+
|-
-
|-
+
|2.5||3.0||0.5||23.4||0.3||0.3||30.0
-
|5||1.5||3||9.5
+
|}
 +
====Liquid Culture====  
 +
J23113(backbone J61002)
 +
 
 +
==August 1==  
 +
====Restriction Enzyme Processing====  
 +
{|class="wikitable"  
 +
!LacP+kil+DT ||BSA||10×H Buffer||EcoR1||Pst1||MilliQ||Total
 +
|-
 +
|5.0||0.5||3.0||0.5||0.5||20.5||30.0
 +
|}37℃ 2h
 +
{|class="wikitable"
 +
!LacP middle copy ||BSA||10×H Buffer||EcoR1||Pst1||MilliQ||Total
 +
|-
 +
|10.0||0.5||3.0||0.5||0.5||15.5||30.0
 +
|}37℃ 2h
 +
====MIniprep====
 +
J23113(backbone J61002)
 +
====Liquid culture====
 +
J23113(backboneJ61002)
 +
Three test tubes of 4 mL LB medium with ampicillin
 +
37℃ overnight
 +
 
 +
==August 2==
 +
==August 3==
 +
====Restriction Enzyme processing====
 +
{|class="wikitable"
 +
!pSB4K5||BSA|| 10×H Buffer||EcoR1||Pst1||MilliQ||Total
 +
|-  
 +
|8.0||0.5||3.0||0.5||0.5||17.5||30.0
 +
|}
 +
{|class="wikitable"
 +
!B0034||BSA||10×H Buffer||Spe1||Pst1||MilliQ||Total
 +
|-
 +
|12.0||0.5||3.0||0.5||0.5||13.5||30.0
|}
|}
-
*pspA : 385fmol<br>
+
37℃ 2h
-
*DT : 36fmol<br><br>
+
====DNA purification====
-
====Transformation====
+
====Ligation====  
-
{| class="wikitable"
+
{|class="wikitable"  
-
!J23107-tatABCD||DT (0.1ng/µL)||DT (0.01ng/µL)||pspA-DT||competent cells on 3/15||total
+
!LacP+kil+DT||pSB4K5||ligation high||Total
-
|-
+
|-  
-
|2||0||0||0||20||22
+
|15||15||15||45
-
|-
+
|}16℃ 1h
-
|0||2||0||0||20||22
+
====Miniprep====
-
|-
+
*J23113(backbone J61002) 218.0μg/mL
-
|0||0||2||0||20||22
+
*J23113(backbone J61002) 252.5μg/mL
-
|-
+
*J23113(backbone J61002) 201.0μg/mL
-
|0||0||0||2||20||22
+
*pSB3C5                  45.0μg/ml  1.60  260/280  1.80  260/230
 +
*pSB4C5                  213.0μg/mL  1.77  260/280  4.40  260/230
 +
 
 +
==August 4==
 +
==August 5==
 +
==August 6==
 +
==August 7==
 +
==August 8==
 +
==August 9==
 +
==August 10==
 +
====Ligation====
 +
{|class="wikitable"
 +
!B0034 Spe1 Pst1||GFP+DT Xba1 Pst1||ligation high||Total
 +
|-  
 +
|2||3||4||9
 +
|}16℃ 1h
 +
====Transformation====
 +
*RBS+GFP+DT
 +
*LacP+kil+DT
 +
*RBS（for control）
 +
:To put 2ng DNA in 20μL competent cell and leave it on ice for 30min
 +
:Heat shock for 60s at 42℃ 
 +
:To leave on ice for 2min
 +
:To spread on ampicillin LB plate
 +
 
 +
==August 11==
 +
==August 12==
 +
==August 13==
 +
====Liquid culture====
 +
B0034 3mL ×3 　　　
 +
:37℃　overnight
 +
 
 +
==August 14==
 +
====Miniprep====
 +
:B0034 0μg/mL
 +
:B0034 235.5μg/mL
 +
:B0034 76.5μg/mL
 +
====Colony PCR====
 +
LacP+kil+DT ×5
 +
{|class="wikitable"
 +
!2×Quick Taq||VF||VF||MilliQ||Total
 +
|-  
 +
|25||1||1||23||50
|}
|}
-
====Screening PCR====
+
25cycles <br>
-
Kil, pspA and torA<br>
+
pspA
-
{| class="wikitable"
+
{|class="wikitable"
-
!Quick Taq||Primer-r||Primer-f||MilliQ||total
+
!10×Buffer for KOD-Plus-Ver2||2mM dNTPs||25mM MgSO4||Primer PsPA  f-p||Primer PsPA  r-s||KOD-Plus-||MilliQ||Total
-
|-
+
|-
-
|25||1||1||23||50
+
|5||5||3||1.5||1.5||1||33||50
 +
|}
 +
====Electrophoresis====
 +
[[File:0814.jpg|200px|right]]
 +
=====positive control(GFP+DT)=====
 +
=====negative control(MilliQ)=====
 +
=====① ～⑥ colony PCR LacP+kil+DT=====  
 +
次の写真
 +
=====① 　　　1kb ladder =====
 +
=====② 　　　Positive control (GFP+DT)=====
 +
=====③ 　　　Negative control (MilliQ)=====
 +
=====④ ～⑨　LacP+kil+DT　①～⑥=====
 +
=====⑩～⑫　  pspA 10μL ,6×buffer 2μL=====
 +
=====⑬　　　　1kb ladder=====
 +
 
 +
==August 15==
 +
====Colony PCR====
 +
pspA  
 +
{|class="wikitable"
 +
!2×Quick Taq||pspA r-s||pspA f-p||MilliQ||Total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
94℃, 2min<br>  
 +
94℃, 30sec<br>
 +
56℃, 30sec<br>
 +
68℃, 1min<br>
 +
→35cycles<br><br>
 +
 
 +
==August 16==
 +
==August 17==
 +
====Colony PCR====
 +
pspA
 +
{|class="wikitable"  
 +
!10×Buffer for KOD-Plus-Ver2||2mM dNTPs||25mM MgSO4||Primer PsPA  f||Primer PsPA  r||KOD-Plus-||MilliQ||Total
 +
|-  
 +
|5||3||3||1.5||1.5||1||35||50  
 +
|-
 +
|5||3||5||1.5||1.5||1||33||50
 +
|}
 +
negative control(MilliQ 50μL)
 +
94℃, 2min<br>
 +
94℃, 15sec<br>
 +
55℃, 30sec<br>
 +
68℃, 1min<br>
 +
→35cycles<br><br>
 +
 
 +
====Electrophoresis====
 +
[[File:0820.JPG|300px|thumb|right]]
 +
 
 +
=====①1kb ladder=====
 +
=====②, ③　　MgSO4 3μL, pspA=====
 +
=====④, ⑤　　MgSO4 5μL, pspA=====
 +
=====⑥      negative control(MilliQ)=====
 +
=====⑧      1kb ladder=====
 +
<br><br><br><br><br>
 +
==August 18==
 +
==August 19==
 +
==August 20==
 +
====Restriction====
 +
{| class="wikitable"
 +
!J23113(201.0ng/μL)||Xba1||Pst1||BufferM||BSA||MilliQ||total
 +
|-
 +
|10||1||1||3||0.5||14.5||30
 +
|-
|}
|}
-
==March 16==
+
==August 21==
-
====Miniprep====
+
====Colony PCR====  
-
torA (pSB1C3) 68.8ng/µL<br>
+
pspA
-
Kil (pSB4K5)  92.7ng/µL<br>
+
{|class="wikitable"
-
torA was red for some reason. We do not know why.<br><br>
+
!10×Buffer||2mM dNTPs||25mM MgSO4||Primer PsPA f||Primer PsPA  r||KOD Plus Neo||MilliQ||Total
-
====Colony PCR====
+
|-
-
{| class="wikitable"
+
|5||3||5||1.5||1.5||1||33||50
-
!Quick Taq||Primer-r||Primer-f||MilliQ||total
+
|-
-
|-
+
|5||3||7||1.5||1.5||1||31||50
-
|25||1||1||23||50
+
|-
-
|}
+
|5||3||10||1.5||1.5||1||28||50
-
94℃, 2min<br>
+
|}
-
94℃, 30sec<br>
+
94℃, 2min<br>  
-
55℃, 30sec<br>
+
98℃, 10sec<br>  
-
68℃, 6sec<br>
+
60℃, 30sec<br>  
 +
68℃, 30sec<br>  
 +
→25cycles<br><br>
 +
====Restriction====
 +
{|class="wikitable"
 +
!J23113(218ng/μL)||Spe1||Pst1||BufferH||BSA||MilliQ||total
 +
|-
 +
|10||1||1||3||0.5||14.5||30
 +
|}
 +
{|class="wikitable"
 +
!torA-GFP-DT(20ng/μL)||Xba1||Pst1||BufferM||BSA||MilliQ||total
 +
|-
 +
|5||0.6||0.6||3||0.5||20.3||30
 +
|}
 +
====Electrophoresis====
 +
 
 +
[[File:0821e.jpg|300px|thumb|right]]
 +
 
 +
=====① 　　　1kb ladder=====
 +
=====②, ③　　torA-GFP-DT(Xba1,Pst1)=====
 +
=====④, ⑤　　J23113(Spe1,Pst1)=====
 +
 
 +
====Transformation====
 +
*LacP-torA-GFP-DT(Backbone pSB3C5)
 +
*BBa_K11704(Backbone pSB1A2)
 +
====Colony PCR====  
 +
 
 +
[[File:0821r.jpg|300px|thumb|right]]
 +
*pspA
 +
{|class="wikitable"  
 +
!10×Buffer||2mM dNTPs||25mM MgSO4||Primer PsPA f||Primer PsPA r||KOD Plus Neo||MilliQ||Total
 +
|-  
 +
|5||5||3||1.5||1.5||1||33||50
 +
|}
 +
{|class="wikitable"
 +
!10×Buffer||2mM dNTPs||25mM MgSO4||Primer PsPA f-p||Primer PsPA r-s||KOD Plus Neo||MilliQ||Total
 +
|-  
 +
|5||5||3||1.5||1.5||1||33||50
 +
|}
 +
*negative control for pspA
 +
We did PCR without a template.<br>
 +
*GFP-DT
 +
{|class="wikitable"
 +
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||Total
 +
|-
 +
|5||5||3||1.5||1.5||1||33||50  
 +
|}  
 +
94℃, 2min<br>  
 +
98℃, 10sec<br>
 +
60℃, 30sec<br>  
 +
68℃, 30sec<br>
 +
 
 +
====Transformation====
 +
*LacP-torA-GFP-DT(Backbone pSB3C5)
 +
*J23107-tatABCD
 +
====PCR====
 +
kil, torA-GFP
 +
{|class="wikitable"
 +
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||DNA||Total
 +
|-
 +
|5||5||3||1.5||1.5||1||32||1||50
 +
|}
 +
94℃, 2min<br>
 +
98℃, 10sec<br>
 +
60℃, 30sec<br>  
 +
68℃, 30sec<br>  
→25cycles<br><br>
→25cycles<br><br>
-
====Electrophoresis====
+
 
-
[[File:Electrophoresis0316.JPG|400px|thumb|right]]
+
==August 22==
-
The results were shown as photograph in the right.<br><br>
+
====Restriction====  
-
====Checking Transformation Efficiency====
+
{| class="wikitable"
-
competent cells that were made on March 15.<br>
+
!pSB3C5||EcoR1||Pst1||BufferH||BSA||MilliQ||total
-
DNA : 0.02ng → 668 colonies  Transformation Efficiency : 3.3×10^7<br>
+
|-
-
DNA : 0.2ng → 1739 colonies  Transformation Efficiency : 8.7×10^6<br><br>
+
|13.3||0.5||0.5||3||0.5||12.2||30
-
====Restriction====
+
|}
-
{| class="wikitable"
+
====Gel extraction====
-
!pSB1C3||EcoR1||Spe1||BSA||BufferM||BufferH||MilliQ||total
+
pSB3C5(EcoR1, Pst1)<br>  
-
|-
+
27.2μg/mL
-
|20||0.2||0.2||0.3||3||0||6.3||30
+
====Transformation(the second time)====  
-
|-
+
*LacP-torA-GFP-DT(Backbone pSB3C5)
-
|5||0.2||0||0.2||0||2||12.6||20
+
*J23107-tatABCD
 +
====Electrophoresis====
 +
写真お願いします。<br>
 +
See "August 21 -Colony PCR-"<br>
 +
1. 1kb ladder<br>  
 +
2. pspA(Primer pspA f&r)<br>
 +
3. negative control for 2<br>  
 +
4. GFP-DT<br>
 +
5. pspA(Primer pspA f-p&r-s)<br>
 +
6. pspA(Primer pspA f-p&r-s)<br>  
 +
7. negative control for 5&6<br>  
 +
====PCR====  
 +
LacP-GFP-DT(a kit of biological parts)
 +
{|class="wikitable"  
 +
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||DNA||Total
 +
|-  
 +
|5||5||3||1.5||1.5||1||32||1||50
 +
|}
 +
====Colony PCR====
 +
pspA
 +
{|class="wikitable"
 +
!10×Buffer||2mM dNTPs||25mM MgSO4||Primer pspA f||Primer pspA r||KOD Plus Neo||MilliQ||Total
 +
|-
 +
|5||5||3||1.5||1.5||1||33||50
 +
|}
 +
{|class="wikitable"
 +
!10×Buffer||2mM dNTPs||25mM MgSO4||Primer pspA f-p||Primer pspA r-s||KOD Plus Neo||MilliQ||Total
 +
|-
 +
|5||5||3||1.5||1.5||1||33||50
 +
|}
 +
94℃, 2min<br>
 +
98℃, 10sec<br>
 +
60℃, 30sec<br>
 +
68℃, 30sec<br>
 +
→30cycles<br>
 +
====Electrophoresis====
 +
写真お願いします。<br>
 +
1. 1kb ladder<br>
 +
2. pspA(Primer pspA f&r)<br>
 +
3. pspA(Primer pspA f-p&r-s)<br>
 +
4. negative control for pspA(Primer pspA f&r)<br>
 +
5. negative control for pspA(Primer pspA f-p&r-s)<br>
 +
====Transformation====
 +
Kil
 +
 
 +
==August 23==
 +
====Colony PCR====
 +
J23107-tatABCD<br>
 +
VF and VR were used for amplifying approximately 2800bp
 +
{|class="wikitable"
 +
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||DNA||Total
 +
|-
 +
|5||5||3||1.5||1.5||1||32.5||0.5||50
 +
|}
 +
94℃, 2min<br>
 +
98℃, 10sec<br>
 +
60℃, 30sec<br>
 +
68℃, 90sec<br>
 +
→25cycles<br>
 +
 
 +
==August 24==
 +
====Purification of PCR products====
 +
pspA(Primer pspA f-p&r-s)<br>
 +
84.1μg/mL<br>
 +
→See "August 22 -Colony PCR-"
 +
====Restriction====
 +
{|class="wikitable"
 +
!pspA||EcoR1||Pst1||BufferH||BSA||MilliQ||total
 +
|-  
 +
|20||0.5||0.5||3||0.5||5.5||30
 +
|}
 +
{|class="wikitable"
 +
!pspA||Xba1||Pst1||BufferM||BSA||MilliQ||total
 +
|-
 +
|20||0.5||0.5||3||0.5||5.5||30
|}
|}
-
at 37℃, for 2 hours.<br>
+
at 37℃ for 1h
-
We did gel extraction for product with EcoR1, Spe1. We got 46.1ng/µL pSB1C3.<br>
+
====Gel extraction====
-
{| class="wikitable"
+
*pspA(EcoR1, Pst1) 30.7μg/mL
-
!Kil(pSB4K5)||EcoR1||Pst1||BSA||BufferH||MilliQ||total
+
*pspA(Xba1, Pst1) 44.0μg/mL
-
|-
+
====Restriction====
-
|5||0.2||0.2||0.2||2||12.4||20
+
{|class="wikitable"  
-
|-
+
!pSB1C3(82.9μg/mL)||EcoR1||Pst1||BufferH||BSA||MilliQ||total  
-
|5||0.2||0||0.2||2||12.6||20
+
|-  
-
|}
+
|20||0.5||0.5||3||0.5||5.5||30
-
for overnight at 37℃.<br>
+
|}  
-
We did this to confirm.<br>
+
→Gel extraction
-
{| class="wikitable"
+
====Electrophoresis====
-
!pspA (pSB1C3)||EcoR1||Pst1||BSA||BufferH||MilliQ||total
+
[[File:0824g.jpg|200px|thumb|right]]<br>  
-
|-
+
1. 1kb ladder<br>  
-
|5||0.2||0.2||0.2||2||12.4||20
+
2. J23107-tatABCD →See "August 23 -PCR-"<br>
-
|-
+
3. negative control for J23107-tatABCD<br>  
-
|5||0.2||0||0.2||2||12.6||20
+
4. pSB1C3(EcoR1, Pst1)<br>
-
|}
+
 
-
for overnight at 37℃.<br>
+
====Ligation====  
-
We did this to confirm.<br><br>
+
{| class="wikitable"  
-
====Ligation====
+
!pSB3C5(EcoR1, Pst1)||pspA(EcoR1, Pst1)||Ligation High Ver.2||total  
-
{| class="wikitable"
+
|-  
-
!torA||pSB1C3||Ligation High Ver.2||total
+
|1||5||3||9  
-
|-
+
|}  
-
|4||2||3||9
+
16℃, overnight
-
|}
+
 
-
{| class="wikitable"
+
==August 25==
-
!MilliQ||pSB1C3||Ligation High Ver.2||total
+
-
|-
+
-
|4||2||3||9
+
-
|}
+
-
at 16℃, for overnight<br>
+
-
*torA→767fmol<br>
+
-
*pSB1C3→68fmol<br>
+
-
==March 17==
+
-
====Miniprep====
+
 
-
J23107-tatABCD 72.7ng/µL<br>
+
   
-
pspA-DT  50.5ng/µL<br><br>
+
 
-
====Checking the Insert====
+
 
-
{| class="wikitable"
+
 
-
!J21037-tatABCD||EcoR1||Pst1||BSA||BufferH||MilliQ||total
+
 
-
|-
+
==August 26==  
-
|5||0.2||0.2||0.2||2||12.4||20
+
-
|-
+
-
|5||0.2||0||0.2||2||12.6||20
+
-
|}
+
-
Success.
+
-
{| class="wikitable"
+
-
!pspA-DT||EcoR1||Pst1||BSA||BufferH||MilliQ||total
+
-
|-
+
-
|5||0.2||0.2||0.2||2||12.4||20
+
-
|-
+
-
|5||0.2||0||0.2||2||12.6||20
+
-
|}
+
-
Failed.
+
 +
 +
-
==March 19==
+
 
 +
 
 +
 
 +
==August 27==  
 +
====Ligation====
 +
{| class="wikitable"
 +
!pSB1C3(EcoR1, Pst1)||pspA(EcoR1, Pst1)||Ligation High Ver.2||total
 +
|-
 +
|1||5||3||9
 +
|}
 +
16℃, overnight
 +
 
 +
====Colony PCR====
 +
Kil (1-7)
 +
{|class="wikitable"
 +
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||Total
 +
|-
 +
|5||5||3||1.5||1.5||1||33||50
 +
|}
 +
94℃, 2min<br>
 +
98℃, 10sec<br>
 +
60℃, 30sec<br>
 +
68℃, 30sec<br>
 +
→30cycles<br>
 +
[[File:0827c.jpg|200px|thumb|right]]
 +
 
 +
====Purification of PCR products====
 +
torA-GFP →See "August 21 -PCR-"
 +
====Restriction====
 +
{| class="wikitable"
 +
!LacP(Backbone:pSB3C5)||Spe1||Pst1||BufferH||BSA||MilliQ||total
 +
|-
 +
|20||0.5||0.5||3||0.5||5.5||30
 +
|}
 +
at 37℃ for 1h<br>
 +
→Gel extraction<br>
 +
{| class="wikitable"
 +
!torA-GFP||Xba1||Pst1||BufferM||BSA||MilliQ||total
 +
|-
 +
|20||0.5||0.5||3||0.5||5.5||30
 +
|}
 +
at 37℃ for 1h<br>
 +
→Purification
 +
====Purification of PCR products====
 +
J23107-tatABCD<br>
 +
42.0μg/mL<br>
 +
→See "August 23 -PCR-"
====Restriction====
====Restriction====
-
{| class="wikitable"
+
{|class="wikitable"  
-
!DT||EcoR1||Xba1||BSA||BufferM||MilliQ||total
+
!J23107-tatABCD||Spe1||EcoR1||BufferM||BSA||MilliQ||total  
-
|-
+
|-  
-
|10||0.2||0.2||0.3||3||16.3||30
+
|20||0.5||0.5||3||0.5||5.5||30  
|}
|}
-
We did Gel extraction and got 17.2ng/µL of DT.<br><br>
+
 
-
====Ligation====
+
==August 28==
-
{| class="wikitable"
+
====Colony PCR====
-
!Kil||DT||Ligation High Ver.2||total
+
Kil (1-16)
-
|-
+
{|class="wikitable"
-
|10||2||6||18
+
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||Total
 +
|-
 +
|5||5||3||1.5||1.5||1||33||50
 +
|}<br>
 +
[[File:0828c.jpg|200px|thumb|right]]
 +
 
 +
====Transformation====
 +
*pspA-pSB1C3
 +
*pspA-pSB3C5
 +
====Liquid culture====
 +
*Kil-3 →Miniprep 100.4μg/mL
 +
*Kil-6
 +
*Kil-7 →Miniprep 77.7μg/mL
 +
*LacP-torA-GFP-1 →Miniprep 88.3μg/mL
 +
*LacP-torA-GFP-2 →Miniprep 85.0μg/mL
 +
*LacP-torA-GFP-3 →Miniprep +++
 +
 
 +
==August 29==
 +
====Kil assay====
 +
evernoteに結果のグラフがあります。
 +
====Liquid culture====
 +
*pspA-pSB1C3(1-3)
 +
*pspA-pSB3C5(1-3)
 +
====Restriction====
 +
{| class="wikitable"
 +
!LacP-Kil-DT(134.7ng/μL)||EcoR1||Spe1||BufferM||MilliQ||total
 +
|-
 +
|10||1||1||3||15||30
 +
|}
 +
37℃, overnight
 +
====Colony PCR====
 +
*LacP-torA-GFP-DT (1-5)
 +
*pspA-pSB1C3 (1-5)
 +
*pspA-pSB3C5 (1-6)
 +
{|class="wikitable"
 +
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||Total
 +
|-
 +
|5||5||3||1.5||1.5||1||33||50
 +
|}
 +
94℃, 2min<br>
 +
98℃, 10sec<br>
 +
60℃, 30sec<br>
 +
68℃, 40sec<br>
 +
→30cycles<br>
 +
====Gel extraction====
 +
J23107-tatABCD(EcoR1, Pst1) -5.9μg/mL
 +
[[File:0829.jpg|none|200px|thumb|right]]
 +
 
 +
==August 30==
 +
====Ligation====
 +
{| class="wikitable"
 +
!J23107-tatABCD(EcoR1, Spe1)22.2μg/mL||DT(EcoR1, Xba1)37.5μg/mL||Ligation High Ver.2||total
 +
|-
 +
|10||1||9||20
 +
|}
 +
at 16℃ for 1h
 +
====Transformation====
 +
J23107-tatABCD-DT (Amp resistance)
 +
====Electrophoresis====
 +
 
 +
[[File:0830e.jpg|none|200px|thumb|right]]<br>
 +
1. LacP-torA-GFP-DT-1<br>  
 +
2. LacP-torA-GFP-DT-2<br>
 +
3. LacP-torA-GFP-DT-3<br>
 +
4. LacP-torA-GFP-DT-4<br>
 +
5. LacP-torA-GFP-DT-5<br>
 +
6. pspA-pSB1C3-1<br>
 +
7. pspA-pSB1C3-2<br>
 +
8. pspA-pSB1C3-3<br>
 +
9. pspA-pSB1C3-4<br>
 +
10.pspA-pSB1C3-5<br>
 +
11.pspA-pSB3C5-1<br>
 +
12.pspA-pSB3C5-2<br>
 +
13.pspA-pSB3C5-3<br>
 +
14.pspA-pSB3C5-4<br>
 +
15.pspA-pSB3C5-5<br>
 +
16.pspA-pSB3C5-6<br>
 +
 
 +
====Miniprep====
 +
*pspA-pSB3C5-1 176μg/mL
 +
*pspA-pSB3C5-2 214μg/mL
 +
*pspA-pSB3C5-3 461μg/mL
 +
*pspA-pSB1C3-2 79μg/mL
 +
====Colony PCR====
 +
LacP-torA-GFP-DT (6-13)
 +
{|class="wikitable"
 +
!2×Quick Taq||VF||VR||MilliQ||Total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
94℃, 2min<br>
 +
94℃, 30sec<br>
 +
56℃, 30sec<br>
 +
68℃, 1min<br>
 +
→30cycles<br><br>
 +
====Restriction====
 +
{| class="wikitable"
 +
!pspA-pSB1C3||EcoR1||Spe1||BufferM||BSA||MilliQ||total
 +
|-
 +
|15||0.5||0.5||5||0.5||28.5||50
 +
|}
 +
at 37℃ for 1h
 +
====Electrophoresis====
 +
[[File:0830r.jpg|none|200px|thumb|right]]<br>
 +
1. 1kb ladder<br>
 +
3. pspA-pSB1C3(EcoR1, Spe1)<br>
 +
 
 +
==August 31==
 +
====Gel extraction====
 +
[[File:0831e.jpg|none|200px|thumb|right]]<br>
 +
pspA-pSB1C3(EcoR1, Spe1) 8.0μg/mL
 +
 
 +
====Purification====
 +
LacP-GFP-DT -0.7μg/mL
 +
====Ligation====  
 +
{| class="wikitable"  
 +
!pspA(EcoR1, Spe1)||DT(EcoR1, Xba1)||Ligation High Ver.2||total
 +
|-
 +
|25||1||13||39
 +
|}
 +
at 16℃ for 1h
 +
====Transformation====
 +
pspA-DT (Amp resistance)
 +
==September 1==
 +
==September 2==
 +
====Colony PCR====
 +
J23107-tatABCD-DT (1-8)
 +
{|class="wikitable"
 +
!2×Quick Taq||VF||VR||MilliQ||Total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
94℃, 2min<br>
 +
94℃, 30sec<br>
 +
55℃, 30sec<br>
 +
68℃, 3min<br>
 +
→25cycles<br><br>
 +
pspA-DT (2-8)
 +
{|class="wikitable"
 +
!2×Quick Taq||VF||VR||MilliQ||Total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
Extension, 10sec<br>
 +
→25cycles<br><br>
 +
====Restriction====
 +
{| class="wikitable"
 +
!LacP-torA-GFP-DT||EcoR1||Spe1||BufferH||MilliQ||total  
 +
|-  
 +
|10||1||1||2||6||20
 +
|}
 +
at 4℃ for overnight
 +
====Electrophoresis====
 +
[[File:0902.jpg|none|200px|thumb|right]]<br>
 +
1. 1kb ladder<br>
 +
3. J23107-tatABCD-DT-1<br>
 +
4. J23107-tatABCD-DT-3<br>
 +
5. J23107-tatABCD-DT-4<br>
 +
6. J23107-tatABCD-DT-5<br>
 +
7. J23107-tatABCD-DT-6<br>
 +
8. J23107-tatABCD-DT-7<br>
 +
9. J23107-tatABCD-DT-8<br>
 +
[[File:0902-2.jpg|none|200px|thumb|right]]<br>
 +
1. 1kb ladder<br>
 +
3. J23107-tatABCD-DT-1<br>
 +
4. J23107-tatABCD-DT-3<br>
 +
5. J23107-tatABCD-DT-4<br>
 +
6. J23107-tatABCD-DT-5<br>
 +
7. J23107-tatABCD-DT-6<br>
 +
8. J23107-tatABCD-DT-7<br>
 +
9. J23107-tatABCD-DT-8<br>
 +
11. LacP-pSB3C5(Spe1, Pst1)
 +
[[File:0902-3.jpg|none|200px|thumb|right]]<br>
 +
1. 1kb ladder<br>
 +
3. pspA-DT-2<br>
 +
4. pspA-DT-3<br>
 +
5. pspA-DT-4<br>
 +
6. pspA-DT-5<br>
 +
7. pspA-DT-6<br>
 +
8. pspA-DT-7<br>
 +
9. pspA-DT-8<br>
 +
 
 +
==September 3==
 +
====PCR====
 +
tatABCD-DT
 +
{|class="wikitable"
 +
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||DNA||Total
 +
|-
 +
|5||5||3||1.5||1.5||1||32||1||50
 +
|}
 +
====Gel extraction====
 +
[[File:0903.jpg|none|200px|thumb|right]]<br>
 +
LacP-torA-GFP-DT →See "September 5 -Gel extraction-"
 +
 
 +
====Restriction====
 +
{| class="wikitable"
 +
!torA-GFP-DT||Xba1||Pst1||BufferM||MilliQ||total
 +
|-
 +
|20||2||2||4||12||40
 +
|}
 +
====Purification====
 +
J23107-tatABCD 29.6μg/mL 1.56(260/280) 1.60(260/230)
 +
====Electrophoresis====
 +
[[File:0903-2.jpg|none|200px|thumb|right]]<br>
 +
1. 1kb ladder<br>
 +
2. J23107-tatABCD<br>
 +
 
 +
====Liquid culture====
 +
*LacP-kil-DT(1)
 +
*pspA-DT(4, 7)
 +
====PCR====
 +
J23107-tatABCD
 +
{|class="wikitable"
 +
!10×Buffer||2mM dNTPs||25mM MgSO4||VF||VR||KOD Plus Neo||MilliQ||DNA||Total
 +
|-
 +
|5||5||3||1.5||1.5||1||29||4||50
 +
|}
 +
94℃, 2min<br>
 +
98℃, 10sec<br>
 +
60℃, 30sec<br>
 +
68℃, 10sec<br>
 +
→30cycles<br><br>
 +
====Gel extraction====
 +
[[File:IMG_2135.jpg|none|200px|thumb|right]]<br>
 +
LacP-torA-GFP-DT 31μg/mL 1.28(260/280) 0.49(260/230)
 +
LacP-torA-GFP-DT 8μg/mL 1.32(260/280) 0.83(260/230)
 +
 
 +
====Transformation====
 +
J23107-tatABCD (Amp resistance)<br>
 +
J23113-kil-DT (Amp resistance)<br>
 +
J23100-kil-DT (CP resistance)<br>
 +
J23101-kil-DT (CP resistance)<br>
 +
J23106-kil-DT (CP resistance)<br>
 +
J23115-kil-DT (CP resistance)→We didn't transform because of lack of culture medium.<br>
 +
J23105-kil-DT (CP resistance)→We didn't transform because of lack of culture medium.<br>
 +
====Electrophoresis====
 +
[[File:IMG_2136.jpg|none|200px|thumb|right]]<br>
 +
1. 1kb ladder<br>
 +
2-7. LacP-torA-GFP-DT<br>
 +
 
 +
====Colony PCR====
 +
LacP-torA-GFP-DT (14-21)
 +
J23113-kil-DT (Amp resistance, 1-4)
 +
J23113-kil-DT (CP resistance, 1-4)
 +
{|class="wikitable"
 +
!2×Quick Taq||primer-f||primer-r||MilliQ||Total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
94℃, 2min<br>
 +
94℃, 30sec<br>
 +
55℃, 30sec<br>
 +
68℃, 1min<br>
 +
→25cycles<br><br>
 +
 
 +
==September 4==
 +
====Liquid culture====
 +
*LacP-kil-DT(4, 5)(Amp resistance)
 +
*pspA-DT(3, 6, 7)(Amp resistance)
 +
====PCR====
 +
We did PCR 10 more cycles to amplify products of PCR that we had done yesterday because we could not amplify J23107-tatABCD on September 3.<br>
 +
94℃, 2min<br>
 +
98℃, 10sec<br>
 +
60℃, 30sec<br>
 +
68℃, 1.5min<br>
 +
→30cycles<br><br>
 +
====Electrophoresis====
 +
[[File:IMG_2137.jpg|none|200px|thumb|right]]<br>
 +
1-8. LacP-torA-GFP-DT<br>
 +
[[File:IMG_2138.jpg|none|200px|thumb|right]]<br>
 +
1-4. J23113-kil-DT (Amp resistance)<br>
 +
5-8. J23113-kil-DT (CP resistance)<br>
 +
 
 +
====Liquid culture====
 +
*LacP-torA-GFP-DT
 +
*J23113-kil-DT
 +
*LacP-kil-DT(with IPTG)
 +
*LacP-kil-DT(without IPTG)
 +
After 20 hour, we quantified OD600.
 +
{|class="wikitable"
 +
!plasmid||OD600
 +
|-
 +
|LacP-kil-DT(with IPTG)||2.116
 +
|-
 +
|LacP-kil-DT(without IPTG)||2.320
 +
|}
 +
We cultured CP tolerance plasmid and give an antibiotic(Amp or Kan or none) after 2 hours.
 +
We quantify OD600.
 +
{|class="wikitable"
 +
!time||1||2||3
 +
|-
 +
|2hours||0.131||0.100||0.606
 +
|-
 +
|add an antibiotic||Amp||Kan||none
 +
|-
 +
|7hours||0.491||0.988||2.634
 +
|-
 +
|19hours||2.253||0.815||2.742
|}
|}
-
We did this for an hour at 16℃.<br><br>
+
 
-
====Restriction====
+
==September 5==
-
{| class="wikitable"
+
====Colony PCR====
-
!GFP||EcoR1||Spe1||BSA||BufferM||MilliQ||total
+
J23107-tatABCD (1-7)
-
|-
+
{|class="wikitable"
-
|10||0.5||0.5||0.5||3||15.5||30
+
!buffer||2mM dNTPs||25mM MgSO4||primer-f||primer-r||KOD Plus Neo||DW||Total
-
|}
+
|-
-
We did this for 4 hours at 37℃<br><br>
+
|5||5||3||1.5||1.5||1||33||50
 +
|}
 +
94℃, 2min<br>
 +
98℃, 10sec<br>
 +
60℃, 30sec<br>
 +
68℃, 1.25min<br>
 +
→30cycles<br><br>
 +
====Gel extraction====
 +
<small>by Kato</small><br>
 +
See "September 3 -Gel extraction-"
 +
LacP-torA-GFP-DT 15.9μg/mL 1.29(260/280) 0.38(260/230)
 +
LacP-torA-GFP-DT 17.2μg/mL 1.23(260/280) 0.48(260/230)
 +
====Miniprep====
 +
*J23113-kil-DT 85.7μg/mL 1.76(260/280) 1.97(260/230)
 +
*LacP-torA-GFP-DT 126.6μg/mL 1.81(260/280) 2.09(260/230)
 +
*LacP-kil-DT-4 62.5μg/mL 1.79(260/280) 1.99(260/230)
 +
*LacP-kil-DT-5 56.0μg/mL 1.79(260/280) 2.01(260/230)
 +
*pspA-DT-3 322μg/mL 1.45(260/280) 1.42(260/230)
 +
*pspA-DT-6 147.6μg/mL 1.25(260/280) 1.37(260/230)
 +
*pspA-DT-7 145.5μg/mL 1.24(260/280) 1.27(260/230)
 +
====Restriction====
 +
{| class="wikitable"
 +
!pspA-DT-6||Xba1||Pst1||BufferM||BSA||MilliQ||total
 +
|-
 +
|20||0.5||0.5||3||0.5||5.5||30
 +
|}
 +
at 37℃ for 1h
 +
====Electrophoresis====
 +
[[File:IMG_2139.jpg|none|200px|thumb|right]]<br>
 +
J23107-tatABCD (1-7)<br>
 +
 
 +
====Colony PCR====
 +
J23107-tatABCD (8-23)
 +
{|class="wikitable"
 +
!2×Quick Taq||VF2||VR||MilliQ||Total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
94℃, 2min<br>
 +
94℃, 30sec<br>
 +
55℃, 30sec<br>
 +
68℃, 1.5min<br>
 +
→30cycles<br><br>
 +
====Gel extraction====
 +
pspA-DT 10.2μg/mL 1.42(260/280) 0.76(260/230)
 +
====Ligation====
 +
{| class="wikitable"
 +
!pspA-DT(Xba1, Pst1)||LacP(pSB3C5)(Spe1, Pst1)||Ligation High Ver.2||total
 +
|-
 +
|5||1||3||9
 +
|}
 +
at 16℃  
 +
====Electrophoresis====
 +
[[File:IMG_2140.jpg|none|200px|thumb|right]]<br>
 +
LacP-torA-GFP-DT(8-15)<br>  
 +
[[File:IMG_2141.jpg|none|200px|thumb|right]]<br>
 +
LacP-torA-GFP-DT(9-23)<br>
 +
 
 +
====Ligation====
 +
{| class="wikitable"
 +
!LacP-torA-GFP-DT(EcoR1, Spe1)||DT 17.2μg/mL(Xba1, Pst1)||Ligation High Ver.2||total
 +
|-
 +
|15||25||20||60
 +
|}
 +
====Liquid culture====
 +
*J23107-tatABCD-8
 +
*J23107-tatABCD-20
 +
*LacP-torA-GFP-DT-21
 +
 
 +
==September 6==
 +
====Miniprep====
 +
*J23107-tatABCD-8 82.4μg/mL 2.06(260/280) 2.95(260/230)
 +
*J23107-tatABCD-20 102.9μg/mL 2.04(260/280) 2.94(260/230)
 +
====Restriction====  
 +
{| class="wikitable"  
 +
!J23107-tatABCD 102.9μg/mL||Spe1||Pst1||BufferH||BSA||MilliQ||total
 +
|-
 +
|10||0.5||0.5||3||0.5||15.5||30
 +
|}
 +
at 37℃ for 1h
 +
====Transformation====
 +
LacP-pspA-DT(pSB3C5)
 +
{|class="wikitable"
 +
!DNA||Competent cell||total
 +
|-
 +
|2||20||22
 +
|}
 +
====Gel extraction====
 +
[[File:IMG_2142.jpg|none|200px|thumb|right]]<br>
 +
J23107-tatABCD(Spe1, Pst1)
 +
→We failed gel extraction because we mistook steps of experiment.
 +
====Electrophoresis====
 +
[[File:IMG_2143.jpg|none|200px|thumb|right]]<br>
 +
LacP-torA-GFP-DT-pspA-DT<br>
 +
====Restriction====
 +
<small>by Terasaka</small><br>
 +
{| class="wikitable"
 +
!J23107-tatABCD||Spe1||Pst1||BufferH||BSA||MilliQ||total
 +
|-
 +
|10||0.5||0.5||3||0.5||15.5||30
 +
|}
 +
====Gel extraction====
 +
[[File:IMG_2144.jpg|none|200px|thumb|right]]<br>
 +
J23107-tatABCD(Spe1, Pst1)
 +
→See "September 7 -Gel extraction-"
 +
 
 +
==September 7==
 +
====Liquid culture====
 +
*LacP-torA-GFP-DT-1 (CP resistance)
 +
*LacP-torA-GFP-DT-21 (CP resistance)
 +
preculture for 1 hour
 +
====Colony PCR====
 +
LacP-torA-GFP-DT (1-8)
 +
LacP-pspA-DT (1-8)
 +
{|class="wikitable"
 +
!2×Quick Taq||VF2||VR||MilliQ||Total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
94℃, 2min<br>
 +
94℃, 30sec<br>
 +
55℃, 30sec<br>
 +
68℃, 1.5min<br>
 +
→30cycles<br><br>
 +
====Restriction====
 +
<small>by Terasaka</small><br>
 +
{| class="wikitable"
 +
!J23107-tatABCD||EcoR1||Spe1||BufferM||BSA||MilliQ||total
 +
|-
 +
|10||0.5||0.5||3||0.5||15.5||30
 +
|}
 +
{| class="wikitable"
 +
!GFP-DT||Xba1||Pst1||BufferM||BSA||MilliQ||total  
 +
|-  
 +
|15||0.5||0.5||3||0.5||10.5||30
 +
|}
 +
====Electrophoresis====
 +
[[File:IMG_2145.jpg|none|200px|thumb|right]]<br>
 +
① -⑧. LacP-pspA-DT<br>
 +
1-8. LacP-torA-GFP-DT<br>
 +
====Gel extraction====
 +
[[File:IMG_2146.jpg|none|200px|thumb|right]]
 +
*J23107-tatABCD(EcoR1, Spe1)
 +
*J23107-tatABCD(Pst1, Spe1) ①57.6μg/mL ②23.4μg/mL
 +
*GFP-DT(Xba1, Pst1)
 +
====Restriction====
 +
{| class="wikitable"
 +
!pspA-DT(145.5μg/mL)||Xba1||Pst1||BufferM||BSA||MilliQ||total
 +
|-
 +
|20||0.5||0.5||3||0.5||5.5||30
 +
|}
 +
37℃, overnight
 +
====Liquid culture====
 +
*LacP-torA-GFP-DT-7 (LB 2mL)<br>
 +
+IPTG 0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM, 0.3mM
 +
*LacP-torA-GFP-DT-7 (LB 5mL)
 +
 
 +
==September 8==
 +
====Check the effect of Amp====
 +
We used 5mL of the liquid culture medium with LacP-torA-GFP-DT-7.<br>
 +
→See "September 7 -Liquid culture-"<br>
 +
We diluted it with LB until OD600=0.444, and dispensed it.<br>
 +
The dispense volume was 1.3mL.<br>
 +
We added 0/13/130μL of Amp(50mg/mL) to it.<br>
 +
=====5.5h after incubating at 37℃=====
 +
{| class="wikitable"
 +
!Amp||0μL||13μL||130μL
 +
|-
 +
|OD600||2.457||2.696||0.310
 +
|}
 +
====Observation through a confocal microscope====
 +
We used the liquid culture media with LacP-torA-GFP-DT + 13μL of Amp(50mg/mL) + IPTG 0/0.1/0.15mM.<br>
 +
Their OD600 was 0.47.<br>
 +
3h after incubating at 37℃, we eliminated each supernatant using a centrifuge, and diluted them with LB to which Amp was added.<br>
 +
2.5h after incubating at 37℃, we observed them through a confocal microscope.<br>
 +
We failed to observe it.
 +
====Gel extraction====
 +
pspA-DT -5.5μg/mL
 +
[[File:IMG_2147.jpg|none|200px|thumb|right]]
 +
====Restriction====
 +
{| class="wikitable"
 +
!pspA-DT-3(322μg/mL)||Xba1||Pst1||BufferM||BSA||MilliQ||total
 +
|-
 +
|10||1||1||3||3||12||30
 +
|}
 +
at 37℃ for 1h
 +
 
 +
==September 9==
 +
====Restriction====
 +
{| class="wikitable"
 +
!J23107-tatABCD(102.9μg/mL)||EcoR1||Spe1||BufferM||MilliQ||total
 +
|-
 +
|10||1||1||3||15||30
 +
|}
 +
====Electrophoresis====
 +
[[File:IMG_2148.jpg|none|200px|thumb|right]]<br>
 +
1. J23107-tatABCD(EcoR1, Spe1)<br>
 +
2. J23107-tatABCD(before restriction)<br>
 +
3. pspA-DT(Xba1, Pst1)<br>
 +
4. pspA-DT(before restriction)<br>
 +
====Gel extraction====
 +
[[File:IMG_2149.jpg|none|200px|thumb|right]]<br>
 +
*J23107-tatABCD(EcoR1, Spe1) 26μg/mL
 +
*pspA-DT(Xba1, Pst1) 199μg/mL
 +
====Liquid culture====
 +
*J23100/J23101/J23106-1
 +
*pspA-DT-5 ×2
 +
*LacP-torA-GFP-DT-21
 +
*GFP generator-2
 +
*LacP-pspA-DT-6
 +
 
 +
==September 10==
 +
====Miniprep====
 +
*J23100-1  105.5μg/mL
 +
*J23101-1  76.0μg/mL
 +
*J23106-1  90.9μg/mL
 +
*pspA-DT-5  ①104.1μg/mL  ②80.0μg/mL
 +
*LacP-torA-GFP-DT-21  79.6μg/mL
 +
*GFP generator-2  84.1μg/mL
 +
*LacP-pspA-DT-6  99.8μg/mL
 +
====Ligation====
 +
*J23107-tatABCD-DT
 +
{| class="wikitable"
 +
!J23107-tatABCD(EcoR1, Spe1)||DT(EcoR1, Xba1)||Ligation High Ver.2||total
 +
|-
 +
|10||5||7.5||22.5
 +
|}
 +
*Negative control for J23107-tatABCD-DT
 +
{| class="wikitable"
 +
!DT(EcoR1, Xba1)||Ligation High Ver.2||total
 +
|-
 +
|5||5||10
 +
|}
 +
*J23107-tatABCD-pspA-DT
 +
{| class="wikitable"
 +
!J23107-tatABCD(Spe1, Pst1)||pspA-DT(Xba1, Pst1)||Ligation High Ver.2||total
 +
|-
 +
|3||6||9||18
 +
|}
 +
*Negative control for J23107-tatABCD-pspA-DT
 +
{| class="wikitable"
 +
!J23107-tatABCD(Spe1, Pst1)||Ligation High Ver.2||total
 +
|-
 +
|3||3||6
 +
|}
 +
====Restriction====
 +
[[File:IMG_2151.jpg|none|200px|thumb|right]]
 +
{| class="wikitable"
 +
!DNA||Spe1||Pst1||BufferH||MilliQ||total
 +
|-
 +
|15||1||1||3||10||30  
 +
|}  
 +
DNA=J23100(105.5μg/mL), J23101(76.0μg/mL), J23106(90.9μg/mL)<br>
 +
at 37℃
 +
====Gel extraction====
 +
[[File:IMG_2152.jpg|none|200px|thumb|right]]
 +
[[File:IMG_2153.jpg|none|200px|thumb|right]]<br>
 +
J23100  27.1μg/mL<br>
 +
J23101  50.2μg/mL<br>
 +
J23106  16.1μg/mL<br>
 +
====Ligation====
 +
*J23100/J23101/J23106-kil-DT
 +
{| class="wikitable"
 +
!J23100/J23101/J23106(Spe1, Pst1)||kil-DT(Xba1, Pst1)||Ligation High Ver.2||total
 +
|-
 +
|1||5||3||9
 +
|}
 +
*Negative control for J23100/J23101/J23106-kil-DT
 +
{| class="wikitable"
 +
!J23100/J23101/J23106(Spe1, Pst1)||MilliQ||Ligation High Ver.2||total
 +
|-
 +
|1||5||3||9
 +
|}
 +
====Transformation====
 +
J23100/J23101/J23106-kil-DT
 +
 
 +
==September 11==
 +
====Colony PCR====
 +
*J23107-tatABCD-DT (1-8)
 +
*J23107-tatABCD-pspA-DT (1-8)
 +
{|class="wikitable"
 +
!2×Quick Taq||VF2||VR||MilliQ||Total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
94℃, 2min<br>
 +
94℃, 30sec<br>
 +
55℃, 30sec<br>
 +
68℃, 3.5min<br>
 +
→30cycles
 +
====Electrophoresis====
 +
[[File:IMG_2155.jpg|none|200px|thumb|right]]
 +
[[File:IMG_2156.jpg|none|200px|thumb|right]]
 +
*J23107-tatABCD-DT (1-8)
 +
*J23107-tatABCD-pspA-DT (1-8)
 +
 
 +
====Restriction====
 +
[[File:IMG_2154.jpg|none|200px|thumb|right]]
 +
{| class="wikitable"
 +
!GFP generator(84.1μg/mL)||Xba1||Pst1||BufferM||BSA||MilliQ||total
 +
|-
 +
|15||1||1||3||3||7||30
 +
|}
 +
{| class="wikitable"
 +
!LacP-torA-GFP-DT(85.0μg/mL)||Spe1||Pst1||BufferH||MilliQ||total
 +
|-
 +
|15||1||1||3||10||30
 +
|}
 +
{| class="wikitable"
 +
!LacP-pspA-DT(99.8μg/mL)||Xba1||Pst1||BufferM||BSA||MilliQ||total
 +
|-
 +
|15||1||1||3||3||7||30
 +
|}
 +
 
 +
====Liquid culture====
 +
pSB1C3(self-ligation) ×5 in LB 3mLCP 3μL<br>
 +
When OD600=approximately 0.6, we added 0/30/60/150/300μL of Amp(50mg/mL) to each liquid culture medium.<br>
 +
1h and 5h after incubating at 37℃, we measured OD600.<br>
 +
{| class="wikitable"
 +
!||1h||5h
 +
|-
 +
|0||-0.020||0.403
 +
|-
 +
|30||-0.046||0.544
 +
|-
 +
|60||0.263||0.007
 +
|-
 +
|150||0.061||0.839
 +
|-
 +
|300||0.093||1.441
 +
|}
 +
====Ligation====
 +
{| class="wikitable"
 +
!pSB3C5(EcoR1, Pst1)||pspA-DT(Xba1, Pst1)||J23107-tatABCD(EcoR1, Spe1)||Ligation High Ver.2||total
 +
|-
 +
|1||3||5||4.5||13.5
 +
|}
 +
4℃, overnight
 +
====Colony PCR====
 +
*J23100-kil-DT (1-6)
 +
*J23101-kil-DT (1-7)
 +
*J23106-kil-DT (1-2)
 +
{|class="wikitable"
 +
!2×Quick Taq||VF||VR||MilliQ||Total
 +
|-
 +
|23||1||1||25||50
 +
|}
 +
====Gel extraction====
 +
[[File:IMG_2159.jpg|none|200px|thumb|right]]
 +
*GFP generator (Xba1, Pst1) 14.1μg/mL
 +
*LacP-torA-GFP-DT (Spe1, Pst1) 42.9μg/mL 1.13(260/280) 1.19(260/230)
 +
*LacP-pspA-DT (Xba1, Pst1) 20.0μg/mL 1.01(260/280) 1.30(260/230)
 +
 
 +
====Electrophoresis====
 +
[[File:IMG_2160.jpg|none|200px|thumb|right]]
 +
*J23107-tatABCD-DT (1-8)
 +
*J23106-kil-DT (1, 2)
 +
*J23100-kil-DT (1-4)
 +
 
 +
====Liquid culture====
 +
*LacP-torA-GFP-DT
 +
*pSB1C3
 +
====Colony PCR====
 +
We did PCR 10 more cycle to amplify products of PCR on 9/11 of J23107-tatABCD-pspA-DT and J23107-tatABCD-DT.
 +
 
 +
 
 +
 
 +
 
 +
 
 +
==September 12==
 +
====Colony PCR====
 +
J23107-tatABCD-pspA-DT (9-15)
 +
94℃, 2min<br>
 +
94℃, 30sec<br>
 +
55℃, 30sec<br>
 +
68℃, 4min<br>
 +
→26cycles<br><br>
 +
====Check the effect of Amp====
 +
We cultured pSB1C3 for overnight.<br>
 +
→See "September 7 -Liquid culture-"<br>
 +
We diluted it with LB, and dispensed it.<br>
 +
We added Amp(50mg/mL) to it until density of Amp was 1/10, 1/30, 1/50 .<br>
 +
{|class="wikitable"
 +
!Amp||start||5 hours  
 +
|-
 +
|1/10||0.557||0.077
 +
|-
 +
|1/30||0.628||0.166
 +
|-
 +
|1/50||0.472||0.055
 +
|-
 +
|0||0.594||2.585
 +
|}
 +
====Electrophoresis====
 +
[[File:IMG_2162.jpg|none|200px|thumb|right]]
 +
*J23107-tatABCD-pspA-DT
 +
[[File:IMG_2161.jpg|none|200px|thumb|right]]
 +
*J23101-kil-DT (1-7)
 +
 
 +
====Restriction====
 +
{| class="wikitable"
 +
!pspA(pSB3C5) (106μg/mL)||EcoR1||Pst1||BufferH||MilliQ||total
 +
|-
 +
|15||1||1||3||10||30
 +
|}
 +
{| class="wikitable"
 +
!pspA-DT-3||Xba1||Pst1||BufferM||BSA||MilliQ||total
 +
|-
 +
|15||1||1||3||3||7||30
 +
|}
 +
{| class="wikitable"
 +
!J23107-tatABCD (102.4μg/mL)||EcoR1||Spe1||BufferM||MilliQ||total
 +
|-
 +
|15||1||1||3||10||30
 +
|}
 +
====Electrophoresis====
 +
[[File:IMG_2165.jpg|none|200px|thumb|right]]
 +
*pspA(pSB3C5)(EcoR1, Pst1)
 +
*pspA-DT(Xba1, Pst1)
 +
*J23107-tatABCD(EcoR1, Spe1)
 +
*pSB1C3(EcoR1, Spe1)
 +
 
 +
====Transformation====
 +
J23107-tatABCD-pspA-DT(pSB3C5)
 +
{|class="wikitable"
 +
!DNA||Competent cell||total
 +
|-
 +
|2||20||22
 +
|}
 +
on ice for 30 minites
 +
heatshock at 42℃ for 1 hour
 +
on ice 2 minites
 +
then add SOC medium 200μl
 +
preculture at 37℃ for 1 hour
 +
incubate CP culture plate
 +
====Gel extraction====
 +
[[File:IMG_2166.jpg|none|200px|thumb|right]]
 +
*pspA(pSB3C5)(EcoR1, Pst1) 26.8μg/mL 1.27(260/280) 0.32(260/230)
 +
*pspA-DT(Xba1, Pst1) 26.1μg/mL 1.13(260/280) 0.55(260/230)
 +
*J23107-tatABCD(EcoR1, Spe1) 1.5μg/mL 1.99(260/280) 0.06(260/230)
 +
 
 +
====Ligation====
 +
{| class="wikitable"
 +
!pSB3C5(EcoR1, Pst1) 26.8μg/mL||pspA-DT(Xba1, Pst1) 26.1μg/mL||J23107-tatABCD(EcoR1, Spe1) 1.5μg/mL||Ligation High Ver.2||total
 +
|-
 +
|1||3||6||5||15
 +
|}
 +
{| class="wikitable"
 +
!pSB3C5(EcoR1, Pst1) 26.8μg/mL||MilliQ||Ligation High Ver.2||total
 +
|-
 +
|1||9||5||15
 +
|}
 +
{| class="wikitable"
 +
!LacP(pSB3C5)(Spe1, Pst1) 10μg/mL||GFP-DT(Xba1, Pst1) 7.0μg/mL||Ligation High Ver.2||total
 +
|-
 +
|1||5||3||9
 +
|}
 +
{| class="wikitable"
 +
!LacP(pSB3C5)(Spe1, Pst1) 10μg/mL||MilliQ||Ligation High Ver.2||total
 +
|-
 +
|1||5||3||9
 +
|}
 +
4℃, overnight
 +
====Observation through a confocal microscope====
 +
Evernoteに上がっている写真をお願いします。<br>
 +
 
 +
==September 13==
 +
 
 +
 +
 
 +
 +
 
 +
 
 +
 
 +
 
 +
====Miniprep====
 +
 
 +
 +
 
 +
 +
 
 +
 
 +
 
 +
 
 +
*J23107-tatABCD-pspA-DT(pSB3C5)-1 130μg/mL 1.37(260/280) 0.79(260/230)
 +
 
 +
 
 +
====Restriction====
 +
{| class="wikitable"
 +
!J23107-tatABCD-pspA-DT(pSB3C5) (130μg/mL)||Spe1||Pst1||BufferH||MilliQ||total
 +
 
 +
 
 +
|-
 +
|10||1||1||3||15||30
 +
 
 +
|}
 +
====Transformation====
 +
 
 +
*LacP-GFP generater
 +
 
 +
*LacP
 +
*J23107-tatABCD-pspA-DT(pSB3C5)
 +
*J23107-tatABCD
 +
====Colony PCR====
 +
 
 +
J23107-tatABCD-pspA-DT<br>  
 +
 
 +
→See "September 14 -Colony PCR-"
 +
 
 +
==September 14==
 +
====Colony PCR====
 +
[[File:IMG_2169.jpg|none|200px|thumb|right]]<br>
 +
See "September 13 -Colony PCR-"<br>
 +
J23107-tatABCD-pspA-DT<br>
 +
 
 +
====Colony PCR====
 +
*J23107-tatABCD-pspA-DT (1-6)
 +
{|class="wikitable"
 +
!2×Quick Taq||VF||VR||MilliQ||Total
 +
|-
 +
|23||1||1||25||50
 +
|}
 +
94℃, 2min<br>
 +
94℃, 30sec<br>
 +
55℃, 30sec<br>
 +
68℃, 4min<br>
 +
→30cycles<br><br>
 +
 
 +
==September 15==
 +
====Liquid culture====
 +
*J23107-tatABCD-pspA-DT-1
 +
*J23107-tatABCD-pspA-DT-2
 +
*J23107-tatABCD-pspA-DT-3
 +
====Miniprep====
 +
*J23107-tatABCD-pspA-DT-1 22.9μg/mL
 +
*J23107-tatABCD-pspA-DT-2 51.8μg/mL 1.57(260/280) 1.01(260/230)
 +
*J23107-tatABCD-pspA-DT-3 28.5μg/mL 1.57(260/280) 0.94(260/230)
 +
====Restriction====
 +
{| class="wikitable"
 +
!J23107-tatABCD-pspA-DT (51.8μg/mL)||EcoR1||Spe1||BufferM||MilliQ||total
 +
|-
 +
|15||0.5||0.5||3||11||30
 +
|}
 +
{| class="wikitable"
 +
!LacP-torA-GFP-DT (79.6μg/mL)||EcoR1||Xba1||BufferM||BSA||MilliQ||total
 +
|-
 +
|10||0.5||0.5||3||0.5||15.5||30
 +
|}
 +
{| class="wikitable"
 +
!LacP-kil-DT (56.0μg/mL)||EcoR1||Xba1||BufferM||BSA||MilliQ||total
 +
|-
 +
|15||0.5||0.5||3||0.5||10.5||30
 +
|}
 +
{| class="wikitable"
 +
!J23107-tatABCD-pspA-DT (51.8μg/mL)||EcoR1||Pst1||BufferM||MilliQ||total
 +
|-
 +
|15||0.5||0.5||3||11||30
 +
|}
 +
37℃, 2 hours
 +
====Electrophoresis====
 +
[[File:IMG_2170.jpg|none|200px|thumb|right]]<br>
 +
①. 1kb ladder<br>
 +
②. J23107-tatABCD-pspA-DT<br>
 +
③. J23107-tatABCD-pspA-DT(EcoR1, Spe1)<br>
 +
④. J23107-tatABCD-pspA-DT(EcoR1, Pst1)<br>
 +
⑤. LacP-kil-DT<br>
 +
⑥. LacP-kil-DT(EcoR1, Xba1)<br>
 +
⑦. LacP-torA-GFP-DT<br>
 +
⑧. LacP-torA-GFP-DT(EcoR1, Xba1)<br>
 +
 
 +
====Gel extraction====
 +
[[File:IMG_2171.jpg|none|200px|thumb|right]]
 +
[[File:IMG_2172.jpg|none|200px|thumb|right]]<br>
 +
①. 1kb ladder<br>
 +
②. J23107-tatABCD-pspA-DT(EcoR1, Spe1)<br>
 +
③. J23107-tatABCD-pspA-DT(EcoR1, Spe1)<br>
 +
④. J23107-tatABCD-pspA-DT(EcoR1, Pst1)<br>
 +
⑤. J23107-tatABCD-pspA-DT(EcoR1, Pst1)<br>
 +
⑥. 1kb ladder<br>
 +
*J23107-tatABCD-pspA-DT(EcoR1, Spe1) 14.2μg/mL 1.28(260/280) 0.54(260/230)
 +
*J23107-tatABCD-pspA-DT(EcoR1, Pst1) 7.3μg/mL 1.55(260/280) 0.38(260/230)
 +
 
 +
====Purification====
 +
*LacP-torA-GFP-DT(EcoR1, Xba1) 7.7μg/mL 1.41(260/280) 0.99(260/230)
 +
*LacP-kil-DT(EcoR1, Xba1) →failed
 +
====Ligation====
 +
{| class="wikitable"
 +
!LacP-torA-GFP-DT(EcoR1, Xba1)||J23107-tatABCD-pspA-DT(EcoR1, Spe1)||Ligation High Ver.2||total
 +
|-
 +
|4||10||7||21
 +
|}
 +
{| class="wikitable"
 +
!pSB1C3(EcoR1, Pst1)||J23107-tatABCD-pspA-DT(EcoR1, Pst1)||Ligation High Ver.2||total
 +
|-
 +
|4||10||7||21
 +
|}
 +
at 16℃ for overnight
 +
 
 +
==September 16==
 +
====Transformation====
 +
LacP-torA-GFP-DT-J23107-tatABCD-pspA-DT<br>
 +
J23107-tatABCD-pspA-DT(pSB1C3)<br>
 +
==September 17==
 +
====Colony PCR====
 +
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (1-6)<br>
 +
J23107-tatABCD-pspA-DT(pSB1C3) (1-6)<br>
 +
J23107-kil-DT (6-10)
 +
====Restriction====
 +
{| class="wikitable"
 +
!LacP-torA-GFP-DT (126μg/mL)||EcoR1||Xba1||BufferM||BSA||MilliQ||total
 +
|-
 +
|10||1||1||3||3||12||30
 +
|}
 +
{| class="wikitable"
 +
!LacP-torA-GFP-DT (126μg/mL)||Xba1||BufferM||BSA||MilliQ||total
 +
|-
 +
|3||1||3||3||20||30
 +
|}
 +
====Electrophoresis====
 +
[[File:IMG_2173.jpg|none|200px|thumb|right]]<br>
 +
A1-A6. J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (1-6)<br>
 +
B1-B6. J23107-tatABCD-pspA-DT(pSB1C3) (1-6)<br>
 +
 
 +
====Liquid culture====
 +
*J23107-tatABCD-pspA-DT(pSB1C3)
 +
*J23107-tatABCD-pspA-DT(pSB3C5)
 +
*LacP-torA-GFP-DT(pSB3C5)
 +
====Electrophoresis====
 +
写真お願いします。<br>
 +
lane1. 1kb ladder<br>
 +
lane2. J23101-kil-DT-6<br>
 +
lane3. J23101-kil-DT-7<br>
 +
lane4. J23101-kil-DT-8<br>
 +
lane5. J23101-kil-DT-10<br>
 +
lane6. LacP-torA-GFP-DT<br>
 +
lane7. LacP-torA-GFP-DT(EcoR1, Xba1)<br>
 +
lane8. LacP-torA-GFP-DT(Xba1)<br>
 +
 
 +
====Miniprep====
 +
*J23106-kil-DT 11.7μg/mL 1.93(260/280) 1.46(260/230)
 +
 
 +
==September 18==
 +
====Gel extraction====
 +
[[File:IMG_2174.jpg|none|200px|thumb|right]]
 +
*LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mL 1.37(260/280) 0.82(260/230)
====Ligation====
====Ligation====
{| class="wikitable"
{| class="wikitable"
-
!pspA||DT||Ligation High Ver.2||total
+
!J23107-tatABCD-pspA-DT(EcoR1, Spe1) 14.2μg/mL||LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mL||Ligation High Ver.2||total
|-
|-
-
|5||5||5||15
+
|8||1.5||9.5||19
|}
|}
{| class="wikitable"
{| class="wikitable"
-
!pspA||pSB1C3||Ligation High Ver.2||total
+
!LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mL||Ligation High Ver.2||total
|-
|-
-
|4||1||3||8
+
|1.5||1.5||3
|}
|}
-
We did these for an hour at 16℃.<br>
+
at 4℃ for 1 hour
-
*pspA (5µL)→377fmol<br>
+
-
*DT→39fmol<br>
+
-
*pspA (4µL)→339fmol<br>
+
-
*pSB1C3→34fmol<br><br>
+
====Transformation====
====Transformation====
-
pspA-DT, pspA (pSB1C3), torA (pSB1C3) and GFP-DT
+
*J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT
 +
*LacP-torA-GFP-DT(EcoR1, Xba1)
 +
====Observation through a confocal microscope====
 +
We made sample for observation through a confocal microscope.<br>
 +
We incubated sample with 0mM/0.5mM IPTG for 1.5 hours.<br>
 +
Then, we incubated onto medium without IPTG for 2.5 hours.<br>
-
==March 20==
 
-
====Screaning PCR====
 
{| class="wikitable"
{| class="wikitable"
-
!Quick Taq||Primer-R||Primer-F||MilliQ||total
+
!IPTG(mM)||medium
|-
|-
-
|25||1||1||23||50
+
|0||LB
-
|}
+
-
*pspA→○<br>
+
-
*pspA-DT→○<br>
+
-
*GFP-DT→○<br>
+
-
*torA→×<br>
+
-
*Kil-DT 6 of 8 sumples→○<br>
+
-
{| class="wikitable"
+
-
!Quick Taq||VR||VF||MilliQ||total
+
|-
|-
-
|25||1||1||23||50
+
|0.1||LB
-
|}
+
-
*pspA→○<br>
+
-
*pspA-DT→×<br><br>
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!torA||EcoR1||Spe1||BSA||BufferM||MilliQ||total
+
|-
|-
-
|10||0.3||0.3||0.3||3||16.1||30
+
|0.1||LB with 0.1mM IPTG  (positive control)
-
|}
+
-
{| class="wikitable"
+
-
!DT||EcoR1||Xba1||BSA||BufferM||MilliQ||total
+
|-
|-
-
|10||0.2||0.2||0.3||3||16.3||30
+
|0||LB with 2 percent glucose
-
|}
+
-
We did this for overnight at 37℃. And we did purification.<br>
+
-
torA  34.2ng/µL<br>
+
-
DT  28.0ng/µL<br><br>
+
-
 
+
-
==March 21==
+
-
====Miniprep====
+
-
GFP-DT-1 : 77.7ng/µL<br>
+
-
GFP-DT-2 : 67.6ng/µL<br><br>
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!pSB1C3||EcoR1||Spe1||BSA||BufferM||MilliQ||total
+
|-
|-
-
|10||0.2||0.2||0.3||3||16.3||30
+
|0.1||LB with 2 percent glucose
|-
|-
-
|5||0.2||0||0.2||2||12.6||20
+
|0||LB with 5 percent glucose
-
|}
+
-
We did this for 3 hours at 37℃, and then we did gel extraction. We got 25.1ng/µL pSB1C3<br><br>
+
-
{| class="wikitable"
+
-
!GFP-DT||EcoR1||Pst1||Xba1||BSA||BufferM||MilliQ||total
+
-
|-
+
-
|5||0.2||0.2||0||0.2||2||12.4||20
+
-
|-
+
-
|5||0.2||0||0||0.2||2||12.6||20
+
-
|-
+
-
|10||0.2||0||0.2||0.3||3||16.3||30
+
-
|}
+
-
We did this for 3 hours at 37℃, and then we did Purification. We got 30.7ng/µL GFP-DT.<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
! ||torA||pSB1C3||pspA||DT||GFP-DT||Ligation High Ver.2||total
+
-
|-
+
-
|1||4||1||0||0||0||3||8
+
|-
|-
-
|2||0||1||7||0||0||4||12
+
|0.1||LB with 5 percent glucose
|-
|-
-
|3||0||0||5||3||0||4||12
+
|0||LB with 2 percent glucose (make medium new after incubate for 1.5 hours)
|-
|-
-
|4||3||0||0||0||5||4||12
+
|0||LB with 2 percent glucose (make medium new after incubate for 1.5 hours)
|}
|}
-
We did this for an hour at16℃.
+
====Restriction====
 +
{| class="wikitable"
 +
!pSB4K5||EcoR1||Pst1||BufferH||MilliQ||total
 +
|-
 +
|10||1||1||3||15||30
 +
|}
 +
{| class="wikitable"
 +
!pSB4K5||EcoR1||BufferH||MilliQ||total
 +
|-
 +
|3||0.5||1||5.5||30
 +
|}
 +
{| class="wikitable"
 +
!pSB4K5||Pst1||BufferH||MilliQ||total
 +
|-
 +
|3||0.5||1||5.5||10
 +
|}
 +
{| class="wikitable"
 +
!LacP-torA-GFP-DT||Xba1||Pst1||BufferM||BSA||MilliQ||total
 +
|-
 +
|10||1||1||3||3||12||30
 +
|}
 +
{| class="wikitable"
 +
!LacP-torA-GFP-DT||Xba1||BufferM||BSA||MilliQ||total
 +
|-
 +
|2||1||1||1||5||10
 +
|}
 +
{| class="wikitable"
 +
!LacP-torA-GFP-DT||Pst1||BufferH||MilliQ||total
 +
|-
 +
|2||1||1||6||10
 +
|}
 +
37℃, 2 hours
 +
====Gel extraction====
 +
*LacP-torA-GFP-DT(Xba1, Pst1) 9.7μg/mL 1.34(260/280) 0.62(260/230)
 +
*pSB4K5(EcoR1, Pst1) 9.6μg/mL 1.28(260/280) 0.32(260/230)
 +
====Miniprep====
 +
*J23107-tatABCD-psp-DT(pSB1C3) 78μg/mL
 +
*J23107-tatABCD-psp-DT(pSB1C3) 48μg/mL
 +
*LacP-torA-GFP-DT 29μg/mL
 +
*J23101-kil-DT 25μg/mL
-
==March 22==
+
====Restriction enzyme processing====  
-
====PCR====
+
-
We did PCR to amplify torA_signal that was product of PCR at March 5 with redesigned primers.<br>
+
-
{| class="wikitable"
+
-
!Buffer||dNTPs||MgSO4||Primer-F||Primer-R||Template||MilliQ||KOD plus neo||total
+
-
|-
+
-
|5||5||3||1.5||1.5||0.5||32.5||1||50
+
-
|}
+
-
94℃, 2min<br>
+
-
98℃, 10sec<br>
+
-
60℃, 30sec<br>
+
-
68℃, 10sec<br>
+
-
→30cycles<br>
+
-
→Purification  110.7ng/µL<br><br>
+
-
====Restriction====
+
-
{| class="wikitable"
+
-
!torA||EcoR1||Spe1||BSA||BufferM||MilliQ||total
+
-
|-
+
-
|10||0.2||0.2||0.3||3||16.3||30
+
-
|}
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
! ||torA||pSSB1C3||pspA||DT||GFP-DT||Ligation high||total
+
-
|-
+
-
|1||4||3||0||0||0||4||11
+
-
|-
+
-
|2||3||0||0||0||3||3||9
+
-
|-
+
-
|3||0||0||5||5||0||5||15
+
-
|}
+
-
*torA (4µL)→512fmol<br>
+
-
*pSB1C3→54fmol<br>
+
-
*torA (3µL)→384fmol<br>
+
-
*GFP-DT→36fmol<br>
+
-
*pspA→377fmol<br>
+
-
*DT→65fmol<br><br>
+
-
==March 23==
 
-
====Screening PCR====
 
-
torA (pSB1C3), torA-GFP-DT and pspA-DT<br>
 
{| class="wikitable"
{| class="wikitable"
-
!Quick Taq||VF||VR||MilliQ||total
+
!J23101-Kil-DT 25µg/ml||BufferM||Xba1||Pst1||BSA||MilliQ||total
-
|-
+
|-
-
|25||1||1||23||50
+
|40||6||1||1||6||6||60
|}
|}
-
E.coli in liquid culture that had pspA(pSB1C3) also expressed RFP.<br><br>
 
-
====Restriction====
 
{| class="wikitable"
{| class="wikitable"
-
!pspA||EcoR1||Spe1||BufferM||BSA||MilliQ||total
+
!J23101-Kil-DT 25µg/ml||BufferM||Xba1||BSA||MilliQ||total
-
|-
+
|-
-
|5||0.3||0.3||3||0.3||21.1||30
+
|3||1||0.5||1||4.5||10
|}
|}
-
And then we did ethanol precipitation<br><br>
 
-
====Ethanol precipitation====
 
-
pspA 11.5ng/µL.<br><br>
 
-
====Miniprep====
 
-
Lacp+pSB3C5-8  77.9µg/mL<br>
 
-
Lacp+pSB3C5-10  69.0µg/mL<br>
 
-
Kil+DT-4  58.0µg/mL<br>
 
-
Kil+DT-7  15.2µg/mL<br><br>
 
-
====Restriction====
 
{| class="wikitable"
{| class="wikitable"
-
!Lacp+pSB3C5-8||Spe1||Pst1||Buffer2||BSA||MilliQ||total
+
!J23101-Kil-DT 25µg/ml||BufferH||Pst1||MilliQ||total
-
|-
+
|-
-
|20||0.5||0.5||3||0.5||5.5||30
+
|3||1||0.5||5.5||60
|}
|}
-
{| class="wikitable"
+
at 37℃ for 2 hours
-
!Kil+DT-4||Xba1||Pst1||Buffer2||BSA||MilliQ||total
+
====Electrophoresis====  
-
|-
+
写真お願いします。<br>
-
|20||0.5||0.5||3||0.5||5.5||30
+
①. pSB4K5<br>
-
|}
+
②. pSB4K5(Pst1)<br>
-
at 37℃ for 1.5 hours<br>
+
③. pSB4K5(EcoR1)<br>
-
And then we did Gel extraction.<br><br>
+
④. pSB4K5(EcoR1, Pst1)<br>
-
====Gel Extraction====
+
⑤. LacP-torA-GFP-DT<br>
-
Lacp+pSB3C5-8  40.4µg/mL<br>
+
⑥. LacP-torA-GFP-DT(Xba1)<br>
-
Kil+DT-4  26.8µg/mL<br>
+
⑦. LacP-torA-GFP-DT(Pst1)<br>
 +
⑧. LacP-torA-GFP-DT(Xba1, Pst1)<br><br>
 +
 
 +
写真お願いします。<br>
 +
①. 1kb ladder<br>
 +
②. J23101-Kil-DT(Xba1, Pst1)<br>
 +
③. J23101-Kil-DT(Xba1, Pst1)<br>  
 +
④. J23101-Kil-DT(Xba1, Pst1)<br>
 +
⑤. J23101-Kil-DT(Xba1, Pst1)<br>
 +
⑥. 1kb ladder<br>
 +
 
 +
 
 +
====Restriction enzyme processing====
-
==March 26==
 
-
====Miniprep====
 
-
torA(pSB1C3) 18.5[ng/µL]<br>
 
-
torA-GFP-DT 20.0[ng/µL]<br><br>
 
-
====Restriction====
 
{| class="wikitable"
{| class="wikitable"
-
!torA(pSB1C3)||EcoR1||Pst1||BufferH||BSA||MilliQ||total
+
!J23101-Kil-DT||BufferM||Xba1||Pst1||BSA||MilliQ||total
-
|-
+
|-
-
|5||0.2||0.2||2||0.2||12.4||20
+
|15||3||1||1||3||7||30
-
|-
+
-
|5||0.2||0||2||0.2||12.6||20
+
-
|}
+
-
{| class="wikitable"
+
-
!torA-GFP-DT||EcoR1||Xba1||Pst1||BufferH||BufferM||BSA||MilliQ||total
+
-
|-
+
-
|5||0.2||0||0.2||2||0||0.2||12.4||20
+
-
|-
+
-
|5||0.2||0||0||2||0||0.2||12.6||20
+
-
|-
+
-
|20||0||0.2||0.2||0||3||0.3||6.3||30
+
-
|}
+
-
We did Gel extraction and then got ??? 28.7[ng/µL]<br><br>
+
-
====Ligation====
+
-
{| class="wikitable"
+
-
!Lacp (pSB3C5)||torA-GFP-DT||Ligation High Ver.2||total
+
-
|-
+
-
|1||5||3||9
+
-
|}
+
-
*Lacp : 22fmol<br>
+
-
*torA-GFP-DT : 197fmol<br>
+
-
{| class="wikitable"
+
-
!pspA||pSB1C3||Ligation High Ver.2||total
+
-
|-
+
-
|10||1||5||16
+
-
|}
+
-
{| class="wikitable"
+
-
!pspA||DT||Ligation High Ver.2||total
+
-
|-
+
-
|10||1||5||16
+
-
|}
+
-
*pspA : 180fmol<br>
+
-
*pSB1C3 : 18fmol<br>
+
-
*DT : 16fmol<br>
+
-
{| class="wikitable"
+
-
!LacP(pSB3C5)||Kil-DT||Ligation High Ver.2||total
+
-
|-
+
-
|1||5||3||9
+
-
|}
+
-
for 2 hours at 16℃<br><br>
+
-
====Transformation====
+
-
{| class="wikitable"
+
-
!Lacp-Kil-DT||competent cell||total
+
-
|-
+
-
|1||10||11
+
|}
|}
-
==March 27==
 
-
====Miniprep====
 
-
We retryed miniprep of torA(pSB1C3).<br>
 
-
We got torA and its concentration was 39.3[ng/µL].<br><br>
 
-
====Transformation====
 
{| class="wikitable"
{| class="wikitable"
-
!Name||Well||Sample||Competent Cells||Total||Plate||Colony
+
!J23101-Kil-DT||BufferM||Xba1||BSA||MilliQ||total
-
|-
+
|-
-
|BBa_K117004 ||14J(2011 plate2)||5||20||?||?||?
+
|3||1||0.5||1||4.5||10
|}
|}
-
We added 100[µL] of culture medium before we started culturing the E.coli.<br><br>
 
-
====Screening PCR====
 
-
{| class="wikitable"
 
-
!Quick Taq||VF2||VR||MilliQ||Total
 
-
|-
 
-
|25||1||1||23||50
 
-
|}
 
-
Lacp-torA-GFP-DT 1, 3, 5, 6, 8, 9 was successful.<br>
 
-
pspA-DT was failed.<br>
 
-
E.coli that had pspA(pSB1C3) did not make any colony.<br>
 
-
Lacp-Kil-DT 1,2,3,4,5,6,7,8 was failed.<br><br>
 
-
====Liquid culture====
 
-
Lacp-torA-GFP-DT<br>
 
-
</div>
 
-
==July 30==
 
-
====Ligation====
 
{| class="wikitable"
{| class="wikitable"
-
!kil+DT(XbaI,PstI)||LacP(SpeI,PstI)||Ligation High ||Total
+
!J23101-Kil-DT||BufferH||Pst1||MilliQ||total
-
|-
+
|-
-
|30||6||36||72
+
|3||1||0.5||5.5||10
-
|}
+
|}<br>
-
==July 31==
+
37℃<br>
-
====Restriction Enzyme Processing ====
+
-
{| class="wikitable"
+
-
!torAGFP+DT ||10×M Buffer||BSA ||MilliQ||XbaI||PstI||Total
+
-
|-
+
-
|2.5||3.0||0.5||23.4||0.3||0.3||30.0
+
-
|}
+
-
====Liquid Culture====
+
-
J23113(backbone J61002)
+
-
==August 1==
+
==September 19==
-
====Restriction Enzyme Processing====
+
====GeneClean II====
-
{|class="wikitable"
+
LacP-torA-GFP-DT<br>
-
!lacP+kil+DT ||BSA||10×H Buffer||EcoRI||PstI||MilliQ||Total
+
4k5
-
|-
+
====Ligation====
-
|5.0||0.5||3.0||0.5||0.5||20.5||30.0
+
{| class="wikitable"
-
|}37℃ 2h
+
!LacP-torA-GFP-DT(Xba1,Pst1) 9.7μg/mL||pSB4K5(EcoR1,Pst1) 9.6μg/mL||J23107-tatABCD-PsPA-DT(EcoR1,Spe1) 14.2μg/mL||Ligation High Ver.2||total
-
{|class="wikitable"
+
|-
-
!lacP middle copy ||BSA||10×H Buffer||EcoRI||PstI||MilliQ||Total
+
|7||2||7||8||24μL
-
|-
+
|}
-
|10.0||0.5||3.0||0.5||0.5||15.5||30.0
+
{| class="wikitable"
-
|}37℃ 2h
+
!pSB4K5(EcoR1,Pst1) 9.6μg/mL||Ligation High Ver.2||MilliQ||total
 +
|-
 +
|2||1||21||24μL
 +
|}<br>
 +
incubate at 37℃ for 2h
 +
====Electrophoresis====
 +
J23101-kil-DT(Xba1,Pst1) (Xba1) (Pst1)<br>
 +
Lane1: 1kb ladder<br>
 +
Lane2: empty<br>
 +
Lane3: plasmid<br>
 +
Lane4: Xba1<br>
 +
Lane5: Pst1<br>
 +
Lane6: Xba1,Pst1<br>
 +
====Liquid Culture====
 +
We did liquid culture J23106-kil-DT(9/10, Amp)'s 6,7 at LB mediumAmp.
 +
====Transformation====
 +
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT<br>
 +
pSB4K5 negative control<br>
 +
====Liquid culture====
 +
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT:1~5<br>
 +
====Colony PCR====
 +
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT<br>
 +
{|class="wikitable"
 +
!VF2||VR||Quick tag||MilliQ||total
 +
|-
 +
|1||1||25||23||50
 +
|}
 +
Predenature 94℃  2min<br>
 +
Denature    94℃  30sec<br>
 +
Annealing  55℃ 30sec<br>
 +
Extension  68℃  5min<br>
 +
→30cycles<br><br>
 +
====Restriction enzyme processing====  
 +
{|class="wikitable"  
 +
!LacP-kil-DT(4) 62.5μg/mL||Xba1||Pst1||10xBSA||MilliQ||BufferM||total
 +
|-
 +
|10||1||1||3||12||3||30
 +
|}
 +
{|class="wikitable"
 +
!LacP-kil-DT(4)||EcoR1||Pst1||MilliQ||BufferH||total
 +
|-  
 +
|10||1||1||15||3||30
 +
|}
 +
{|class="wikitable"
 +
!LacP-kil-DT||Xba1||10xBSA||MilliQ||BufferM||total
 +
|-
 +
|2||0.5||1||5.5||1||10
 +
|}
 +
{|class="wikitable"
 +
!LacP-kil-DT||EcoR1||MilliQ||BufferH||total
 +
|-
 +
|2||0.5||6.5||1||10
 +
|}
 +
{|class="wikitable"
 +
!LacP-kil-DT||Pst1||MilliQ||BufferH||total
 +
|-
 +
|2||0.5||6.5||1||10
 +
|}  
 +
at 37℃, 2 hours
 +
====Electrophoresis====
 +
Lane1: 1kb ladder<br>
 +
Lane2: empty<br>
 +
Lane3: plasmid<br>
 +
Lane4: EcoR1<br>
 +
Lane5: Xba1<br>
 +
Lane6: Pst1<br>
 +
Lane7: EcoR1,Pst1<br>
 +
Lane8: Xba1,Pst1<br>
 +
====Electrophoresis====
 +
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT<br>
 +
Lane1: ladder<br>
 +
Lane2: 1<br>
 +
Lane3: 2<br>
 +
Lane4: 3<br>
 +
Lane5: 4<br>
 +
Lane6: 5<br>
 +
====Ligation====
 +
{| class="wikitable"  
 +
!LacP-torA-GFP-DT(Xba1,Pst1) 9.7μg/mL||pSB4K5(EcoR1,Pst1) 9.6μg/mL||J23107-tatABCD-PsPA-DT(EcoR1,Spe1) 14.2μg/mL||Ligation High Ver.2||total
 +
|-
 +
|7||4||7||9||27μL
 +
|}<br>
 +
(Negative control)<br>
 +
{| class="wikitable"
 +
!pSB4K5(EcoR1,Pst1) 9.6μg/mL||Ligation High Ver.2||MilliQ||total
 +
|-
 +
|4||9||14||27μL
 +
|}<br>
 +
====Restriction enzyme processing====
 +
{|class="wikitable"
 +
!torA-R9 101.8μg/mL||bufferH||MilliQ||EcoR1||Pst1||total
 +
|-  
 +
|10||3||15||1||1||30
 +
|}
 +
{|class="wikitable"
 +
!torA-R9 101.8μg/mL||bufferH||MilliQ||EcoR1||Pst1||total
 +
|-
 +
|3||1||5.5||0.5||0||10
 +
|}
 +
{|class="wikitable"
 +
!torA-R9 101.8μg/mL||bufferH||MilliQ||EcoR1||Pst1||total
 +
|-
 +
|3||1||5.5||0||0.5||10
 +
|}
 +
====PCR====
 +
{|class="wikitable"
 +
!torA-R9||MilliQ||10xPCR buffer for KOD plus neo||2mM dNTP3||25mM MgSO4||M13||KOD plus neo||total
 +
|-
 +
|0.5||34||5||5||3||1.5||1||50
 +
|}  
 +
Predenature 94℃  2min<br>
 +
Denature    98℃  10sec<br>
 +
Annealing  60℃ 30sec<br>
 +
Extension  68℃ 20sec<br>
 +
→35cycles<br><br>
 +
====Electrophoresis====
 +
torA-R9,plasmid,(EcoR1),(Pst1),(EcoR1,Pst1)<br>
 +
Lane1: 1kb ladder<br>
 +
Lane2: empty<br>
 +
Lane3: plasmid<br>
 +
Lane4: EcoR1<br>
 +
Lane5: Pst1<br>
 +
Lane6: EcoR1,Pst1<br>
 +
Lane7: empty<br>
 +
Lane8: 1kb ladder<br>
 +
====Liquid culture====
 +
LacP-tatA-GFP-DT (9/4)
 +
====PCR purification====
 +
torA-R9 68.0μg/mL
-
====MIniprep====
+
==September 20==  
-
J23113(backbone J61002)
+
====Gel extraction====  
-
====Liquid culture====
+
LacP-kil-DT (EcoR1,Pst1) (Xba1,Pst1)<br>
-
J23113(backboneJ61002)
+
====Restriction enzyme processing====
-
Three test tubes of 4 mL LB medium with ampicillin
+
{|class="wikitable"
-
37℃ overnight
+
!torA-R9 68.0μg/mL||bufferM||Spe1||MilliQ||Total
-
==August 2==
+
|-
-
==August 3==  
+
|20||3||1||6||30
-
====Restriction Enzyme processing====
+
|}
 +
at 37℃, 2h
 +
====Ligation====
 +
{| class="wikitable"
 +
!LacP-kil-DT(Xba1,Pst1) 12.4μg/mL||pSB1C3(EcoR1,Pst1) 34.2μg/mL||J23107-tatABCD-PsPA-DT(EcoR1,Spe1) 14.2μg/mL||Ligation High Ver.2||total
 +
|-
 +
|7||4||7||9||27μL
 +
|} 16℃, 1h<br>
 +
(Negative control)<br>
 +
{| class="wikitable"
 +
!pSB1C3(EcoR1,Pst1) ||Ligation High Ver.2||MilliQ||total
 +
|-
 +
|4||9||14||27μL
 +
|}<br>
 +
====Transformation====
 +
Using above Ligation and Negative control's product and torA-R9.<br>
 +
====Miniprep====
 +
J23106-kil-DT 6;  12.0μg/mL, (260/280): 1.78, (260/230): 1.73<br>
 +
J23106-kil-DT 7;  13.6μg/mL, (260/280): 1.85, (260/230): 1.73<br>
 +
====Restriction enzyme processing====
 +
{|class="wikitable"
 +
!GFP-DT 84.1μg/mL||bufferM||10xBSA||BbaI||MilliQ||total
 +
|-
 +
|15||3||3||1||8||30
 +
|}
 +
at 37℃, 2 hours
 +
====Colony PCR====
 +
We made J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT's 1~8 samples.
 +
{|class="wikitable"
 +
!Quick tag||tat f primer||VR||MilliQ||total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
Predenature 94℃  2min<br>
 +
Denature    94℃  30sec<br>
 +
Annealing  54℃ 30sec<br>
 +
Extension  68℃ 4min<br>
 +
→30cycles<br>
 +
====Liquid culture====  
 +
We did liquid culture  J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT's 1~4 samples.
 +
====Electrophoresis====
 +
J23701-tatABCD-pspA-DT-LacP-torA-GFP-DT
 +
Lane1: 1kb ladder<br>
 +
Lane2: 1<br>
 +
Lane3: 2<br>
 +
Lane4: 3<br>
 +
Lane5: 4<br>
 +
Lane6: 5<br>
 +
Lane7: 6<br>
 +
Lane8: 7<br>
 +
Lane9: 8<br>
 +
Lane10: 1kb ladder<br>
 +
Lane11: empty<br>
 +
Lane12: empty<br>
 +
torA-R9 (EcoR1,Pst1): 31μg/mL, (280/280): 0.80, (280/230): 0.39<br>
 +
pSB1C3(EcoR1,Pst1): 22μg/mL, (280/280):1.33, (280/230):0.6<br>
 +
torA-R9 (Spe1): 56μg/mL, (280/280): 1.11, (280/230): 0.69<br>
 +
GFP-DT(Xba1): 45μg/mL, (280/280): 1.15, (280/230): 0.88<br>
 +
====Ligation====
 +
{| class="wikitable"
 +
!torA-R9 (EcoR1,Pst1)||pSB1C3(EcoR1,Pst1)||Ligation High Ver.2||total
 +
|-
 +
|8||2||5||15μL
 +
|}
 +
{| class="wikitable"
 +
!torA-R9 (Spe1)||pSB1C3(Xba1)||Ligation High Ver.2||total
 +
|-
 +
|3||3||3||9μL
 +
|}
 +
at 16℃, 1hour
 +
====Colony PCR====
 +
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (4k5)<br>
 +
numbering 9~15
 +
====Transformation====
 +
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (4k5)<br>
 +
J23107-tatABCD-pspA-DT-LacP-torA-kil-DT(pSB1C3) <br>
 +
Negative control (pSB1C3)<br>
 +
Negative control(pSB4K5)<br>
 +
torA-R9(pSB1C3)<br>
 +
====Liquid culture====
 +
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT: colony PCR(9~15)<br>
 +
{|class="wikitable"
 +
!2xQuick tag||VF||VR||MilliQ||total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
Predenature 94℃  2min<br>
 +
Denature    94℃  30sec<br>
 +
Annealing  55℃ 30sec<br>
 +
Extension  68℃ 5min<br>
 +
→30cycles<br>
 +
====PCR====
 +
{|class="wikitable"
 +
!torA-R9-GFP-DT||MilliQ||KOD plus ver.2 buffer||dNTP3||MgSO4||M13||VR||KOD plus||total
 +
|-
 +
|1||35||5||5||2||1.5||1.5||1||52
 +
|}
 +
Predenature 94℃  2min<br>
 +
Denature    94℃  15sec<br>
 +
Annealing  55℃ 30sec<br>
 +
Extension  68℃ 5min<br>
 +
→30cycles<br>
 +
====Miniprep====
 +
J23701-tatABCD-pspA-DT-LacP-torA-GFP-DT:2,3<br>
 +
2: 10.4μg/mL, (250/280): 1.45, (260/230): 0.95<br>
 +
3: 6.0μg/mL, (250/280): 1.32, (260/230): 0.79<br>
 +
(*)100μL
 +
=====Electrophoresis=====
 +
torA-R9-GFP-DT(after PCR)
 +
 
 +
==September 21==
 +
====Gel extraction====
 +
torA-R9-GFP-DT(after PCR): 10.3μg/mL, (260/280): 1.28, (220/230): 0.49<br>
 +
====Electrophoresis====
 +
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(kan): 9~15<br>
 +
====Electrophoresis====
 +
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT: 1,2<br>
 +
Lane1: empty<br>
 +
Lane2: empty<br>
 +
Lane3: 2<br>
 +
Lane4: 3<br>
 +
Lane5: 1kb ladder<br>
 +
====Restriction enzyme processing====
 +
{|class="wikitable"
 +
!LacP-kil-DT(3)||Xba1||Pst1||10xBSA||MilliQ||BufferM||total
 +
|-
 +
|15||1||1||3||7||3||30
 +
|}
 +
{|class="wikitable"
 +
!LacP-torA-GFP-DP(9/5)||Xba1||Pst1||10xBSA||MilliQ||BufferM||total
 +
|-
 +
|15||1||1||3||7||3||30
 +
|}
 +
{|class="wikitable"
 +
!LacP-kil-DT||Xba1||10xBSA||MilliQ||BufferM||total
 +
|-
 +
|2||0.5||1||5.5||1||10
 +
|}
 +
{|class="wikitable"
 +
!LacP-torA-GFP-DP||Xba1||10xBSA||MilliQ||BufferM||total
 +
|-
 +
|2||0.5||1||5.5||1||10
 +
|}
 +
{|class="wikitable"
 +
!LacP-kil-DT||Pst1||MilliQ||BufferH||total
 +
|-
 +
|2||0.5||6.5||1||10
 +
|}
 +
{|class="wikitable"
 +
!LacP-torA-GFP-DP||Xba1||Pst1||MilliQ||BufferH||total
 +
|-
 +
|2||0.5||1||6.5||1||10
 +
|}
 +
{|class="wikitable"
 +
!J23107-tatABCD-pspA-DT||Spe1||Pst1||BufferH||MilliQ||total
 +
|-
 +
|20||1||1||4||14||40
 +
|}
 +
{|class="wikitable"
 +
!J23107-tatABCD-pspA-DT||Spe1||BufferM||MilliQ||total
 +
|-
 +
|2||0.5||1||6.5||10
 +
|}
 +
{|class="wikitable"
 +
!J23107-tatABCD-pspA-DT||Pst1||BufferH||MilliQ||total
 +
|-
 +
|2||0.5||1||6.5||10
 +
|}<br>
 +
at 37℃, 2hours
 +
 
 +
==September 22==
 +
====Miniprep====
 +
torA-R9(Amp)1: 113.7μg/mL, (260/280): 1.96, (280/230): 2.42<br>
 +
torA-R9(Amp)2:  97.8μg/mL, (260/280): 1.67, (280/230): 1.72<br>
 +
torA-R9(Amp)3: 104.2μg/mL, (260/280): 1.93, (280/230): 2.67<br>
 +
LacP-torA-GFP-DT 6: 37.4μg/mL, (260/280): 2.24, (280/230): 3.27<br>
 +
 
 +
====Electrophoresis====
 +
LaneA: J23107-tatABCD (9/6)<br>
 +
LaneB: J23107-tatABCD-pspA-DT pSB3C5 (9/13)<br>
 +
LaneC: J23107-tatABCD-pspA-DT 1C5 (9/15)<br>
 +
LaneD: J23107-tatABCD-pspA-DT (9/15)<br>
 +
LaneE: J23107-tatABCD-pspA-DT(pSB1C3) (9/18)<br>
 +
LaneF: J23107-tatABCD-pspA-DT(pSB3C5) (9/18)<br>
 +
LaneG: J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT:2 (9/20)<br>
 +
LaneH: J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT:3 (9/20)<br>
 +
LaneA~H are (EcoR1,Pst1)<br>
 +
====Restriction enzyme processing====
 +
{|class="wikitable"
 +
!J23107-tatABCD-pspA-DT-LacPtorA-GFP-DT:2||BufferH||Pst1||MilliQ||total
 +
|-
 +
|15||3||1||12||30
 +
|}
 +
{|class="wikitable"
 +
!J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT:3||BufferH||Pst1||MilliQ||total
 +
|-
 +
|15||3||1||12||30
 +
|}<br>
 +
====Restriction enzyme processing====
 +
{|class="wikitable"
 +
!LacP-torA-GFP-DT(37.4μg/mL)||BufferH||EcoR1||Pst1||total
 +
|-
 +
|25||3||1||1||30
 +
|}<br>
 +
====Electrophoresis====
 +
1: J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT:2<br>
 +
2: LacP-torA-GFP-DT (EcoR1,Pst1) (9/22)<br>
 +
3: J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT:3<br>
 +
 
 +
==September 23==
 +
====Electrophoresis====
 +
写真お願いします。
 +
*LacP-torA-GFP-DT(EcoR1, Pst1)
 +
lane_1 1kb ladder
 +
lane_3 DNA
 +
lane_4 DNA
 +
lane_5 DNA
 +
====Gel extraction====
 +
写真お願いします。
 +
*J23107-tatABCD-pspA-DT(pSB1C3)(Spe1, Pst1)
 +
*LacP-torA-GFP-DT(Xba1, Pst1)
 +
lane_1 1kb ladder <br>
 +
lane_3 J23107-tatABCD-pspA-DT(pSB1C3)(Spe1, Pst1) <br>
 +
lane_5 LacP-torA-GFP-DT(Xba1, Pst1)<br>
 +
 
 +
====Restriction====
 +
{|class="wikitable"
 +
!LacP-torA-GFP-DT (78.4μg/mL)||Xba1||Pst1||BufferM||BSA||MilliQ||total
 +
|-
 +
|15||1||1||3||3||7||30
 +
|}
 +
{|class="wikitable"
 +
!LacP-torA-GFP-DT (78.4μg/mL)||Xba1||BufferM||BSA||MilliQ||total
 +
|-
 +
|3||0.5||1||1||4.5||10
 +
|}
 +
{|class="wikitable"
 +
!J23107-tatABCD-pspA-DT(pSB1C3) (78μg/mL)||Spe1||Pst1||BufferH||MilliQ||total
 +
|-
 +
|9||1||1||2||7||20
 +
|}
 +
====Ligation====
 +
{| class="wikitable"
 +
!LacP-torA-GFP-DT(EcoR1, Pst1) (5.5μg/mL)||pSB4K5 (9.6μg/mL)||Ligation High Ver.2||total
 +
|-
 +
|8||4||6||18
 +
|}
 +
at 16℃ for 1 hour<br>
 +
====Liquid culture====
 +
*LacP-tatABCD-pspA-DT (2, 3, 5, 6)
 +
====Colony PCR==== 
 +
LacP-tatABCD-pspA-DT (1-6)
 +
{|class="wikitable"
 +
!2×Quick Taq||VF2||VR||MilliQ||Total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
94℃, 2min<br>
 +
94℃, 30sec<br>
 +
55℃, 30sec<br>
 +
68℃, 4min<br>
 +
→30cycles<br><br>
 +
====Electrophoresis====
 +
写真お願いします。<br>
 +
①, LacP-torA-GFP-DT<br>
 +
②, LacP-torA-GFP-DT(Xba1, Pst1)<br>
 +
③, LacP-torA-GFP-DT(Xba1)<br>
 +
④, LacP-torA-GFP-DT(Pst1)<br>
 +
⑤, J23107-tatABCD-pspA-DT(Spe1, Pst1)<br>
 +
====Liquid culture====
 +
*LacP-torA-GFP-DT (4, 7)
 +
*LacP-GFP generater (1-3)
 +
*T7-6His-GFP-DT (1, 2)
 +
====Gel extraction====
 +
写真お願いします。
 +
*LacP-torA-GFP-DT (Xba1, Pst1) 6.8μg/mL 1.27(260/280) 0.40(260/230)
 +
====Purification====
 +
*J23107-tatABCD-pspA-DT(Spe1, Pst1) 15.1μg/mL 1.49(260/280) 0.80(260/230)
 +
====Ligation====
 +
{| class="wikitable"
 +
!J23107-tatABCD-pspA-DT(Spe1, Pst1) (15.1μg/mL)||LacP-torA-GFP-DT (Xba1, Pst1) (6.8μg/mL)||Ligation High Ver.2||total
 +
|-
 +
|10||4||7||21
 +
|}
 +
{| class="wikitable"
 +
!J23107-tatABCD-pspA-DT(Spe1, Pst1) (15.1μg/mL)||LacP-kil-DT (Xba1, Pst1) (12.4μg/mL)||Ligation High Ver.2||total
 +
|-
 +
|8||2||5||15
 +
|}
 +
at 16℃ for 1 hour<br>
 +
====Transformation====
 +
LacP-torA-GFP-DT(pSB4K5)<br>
 +
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT<br>
 +
J23107-tatABCD-pspA-DT-LacP-kil-DT<br>
 +
{| class="wikitable"
 +
!DNA||competent cell||total
 +
|-
 +
|2||10||12
 +
|}
 +
preculture at 37℃ for 1 hour
 +
culture at 37℃ for 16 hours
 +
====Electrophoresis====
 +
写真お願いします。<br>
 +
J23107-tatABCD-pspA-DT(pSB1C3) (1-6)<br>
 +
lane_1 1kb ladder
 +
lane_2~7 DNA
 +
lane_8 1kb ladder
 +
 
 +
==September 24==
 +
====Miniprep====
 +
*J23107-tatABCD-pspA-DT-2 139.4μg/ml  1.69(260/280) 1.83(260/230)
 +
*J23107-tatABCD-pspA-DT-3 134.7μg/ml  1.61(260/280) 1.69(260/230)
 +
*J23107-tatABCD-pspA-DT-5 139.3μg/ml  1.55(260/280) 1.45(260/230)
 +
*J23107-tatABCD-pspA-DT-6 138.5μg/ml  1.58(260/280) 1.40(260/230)
 +
*LacP-torA-GFP-DT-4
 +
*LacP-torA-GFP-DT-7
 +
*T7-6His-GFP-DT-1
 +
*T7-6His-GFP-DT-2
 +
====Colony PCR==== 
 +
LacP-GFP generater (1-3)
 +
{|class="wikitable"
 +
!2×Quick Taq||VF2||VR||MilliQ||Total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
94℃, 2min<br>
 +
94℃, 30sec<br>
 +
55℃, 30sec<br>
 +
68℃, 1min<br>
 +
→30cycles<br><br>
 +
 
 +
写真お願いします。<br>
 +
①-③, LacP-GFP generater (1-3) <br>
 +
 
 +
====Liquid culture====
 +
*J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (1-4)
 +
*J23107-tatABCD-pspA-DT-LacP-kil-DT (1-3)
 +
====Colony PCR==== 
 +
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (1-4) <br>
 +
J23107-tatABCD-pspA-DT-LacP-kil-DT (1-3) <br>
 +
{|class="wikitable"
 +
!2×Quick Taq||VF2||VR||MilliQ||Total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
94℃, 2min<br>
 +
94℃, 30sec<br>
 +
55℃, 30sec<br>
 +
68℃, 5min<br>
 +
→30cycles<br><br>
 +
 
 +
====Electrophoresis====
 +
写真お願いします。<br>
 +
lane1, 1kb ladder<br>
 +
lane2-5, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (1-4)<br>
 +
lane6-8, J23107-tatABCD-pspA-DT-LacP-kil-DT (1-3)<br>
 +
 
 +
==September 25==
 +
====Liquid culture====
 +
*J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB4K5)-2
 +
====Miniprep====
 +
J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3)-3: 26.2μg/mL, (260/280): 1.50, (280/230): 0.65<br>
 +
J23107-tatABCD-pspA-DT-LacP-torA-GFP(pSB1C3)-3: 24.8μg/mL, (260/280): 1.56, (280/230): 0.76<br>
 +
LacP-GFP generater-1: 42.0μg/mL, (260/280): 1.58, (280/230): 0.94<br>
 +
====Restriction enzyme processing====
{|class="wikitable"
{|class="wikitable"
-
!pSB4K5||BSA|| 10×H Buffer||EcoRI||PstI||MilliQ||Total  
+
!J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (26.2ng/µL)!!EcoRl!!Spel!!buffer M!!MiliQ!!Total
|-
|-
-
|8.0||0.5||3.0||0.5||0.5||17.5||30.0
+
|15||1||1||3||10||30
|}
|}
{|class="wikitable"
{|class="wikitable"
-
!B0034||BSA||10×H Buffer||SpeI||PstI||MilliQ||Total
+
!J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (26.2ng/µL)!!EcoRl!!buffer M!!MiliQ!!Total
|-
|-
-
|12.0||0.5||3.0||0.5||0.5||13.5||30.0
+
|2||0.5||1||6.5||10
-
|}37℃ 2h
+
|}
-
====DNA purification====
+
-
====Ligation====
+
{|class="wikitable"
{|class="wikitable"
-
!lacP+kil+DT||pSB4K5||ligation high||Total  
+
!J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (26.2ng/µL)!!Spel!!buffer M!!MiliQ!!Total
|-
|-
-
|15||15||15||45
+
|2||0.5||1||6.5||10
-
|}16℃ 1h
+
|}
-
====Miniprep====
+
-
*J23113(backbone J61002) 218.0μg/mL
+
-
*J23113(backbone J61002) 252.5μg/mL
+
-
*J23113(backbone J61002) 201.0μg/mL
+
-
*pSB3C5                  45.0μg/ml  1.60  260/280  1.80  260/230
+
-
*pSB4C5                  213.0μg/mL  1.77  260/280  4.40  260/230
+
-
==August 4==
+
-
==August 5==
+
-
==August 6==
+
-
==August 7==
+
-
==August 8==
+
-
==August 9==
+
-
==August 10==
+
-
====Ligation====
+
{|class="wikitable"
{|class="wikitable"
-
!B0034 SpeI PstI||GFP+DT XbaI PstI||ligation high||Total
+
!J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (24.8ng/µL)!!EcoRl!!Pstl!!buffer H!!MiliQ!!Total
|-
|-
-
|2||3||4||9
+
|15||1||1||3||10||30
-
|}16℃ 1h
+
|}
-
 
+
-
====Transformation====
+
-
*RBS+GFP+DT
+
-
*lacP+kil+DT
+
-
*RBS（for control）
+
-
:To put 2ng DNA in 20μL competent cell and leave it on ice for 30min
+
-
:Heat shock for 60s at 42℃ 
+
-
:To leave on ice for 2min
+
-
:To spread on ampicillin LB plate
+
-
==August 11==
+
-
==August 12==
+
-
==August 13==
+
-
====Liquid culture====
+
-
B0034 3mL ×3 　　　
+
-
:37℃　overnight
+
-
==August 14==
+
-
====Miniprep====
+
-
:B0034 0μg/mL
+
-
:B0034 235.5μg/mL
+
-
:B0034 76.5μg/mL
+
-
====Colony PCR====
+
-
lacP+kil+DT ×5
+
{|class="wikitable"
{|class="wikitable"
-
!2×Quick Taq||VF||VF||MilliQ||Total
+
!J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (24.8ng/µL)!!Pstl!!buffer H!!MiliQ!!Total
|-
|-
-
|25||1||1||23||50
+
|2||0.5||1||6.5||10
-
|}25cycles
+
|}
-
pspA
+
{|class="wikitable"
{|class="wikitable"
-
!10×Buffer for KOD-Plus-Ver2||2mM dNTPs||25mM MgSO4||Primer PsPA  f-p||Primer PsPA  r-s||KOD-Plus-||MilliQ||Total
+
!J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (24.8ng/µL)!!EcoRl!!buffer H!!MiliQ!!Total
|-
|-
-
|5||5||3||1.5||1.5||1||33||50
+
|2||0.5||1||6.5||10
|}
|}
 +
at 37℃ for 2 hours
 +
====Electrophoresis====
 +
写真お願いします。<br>
 +
lane1, 1kb ladder<br>
 +
lane2, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3)<br>
 +
lane3, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl, Spel)<br>
 +
lane4, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl)<br>
 +
lane5, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (Spel)<br>
 +
lane6, J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3)
 +
lane7, J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (EcoRl, Pstl)<br>
 +
lane8, J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (EcoRl)<br>
 +
lane9, J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (Pstl)<br>
 +
lane10, LacP-torA-GFP-DT(EcoR1,Xba1)(9/15)<br>
 +
lane11, LacP-torA-GFP-DT(EcoR1,Xba1)(9/17)<br>
 +
lane12, 1kb ladder<br>
-
====Electrophoresis====
+
→The bands didn't appear, so we did one more electrophoresis.<br>
-
写真貼ってください
+
写真お願いします。<br>
-
=====positive control(GFP+DT)=====
+
lane1, 1kb ladder<br>
-
=====negative control(MilliQ)=====
+
lane2, LacP-torA-GFP-DT(EcoR1,Xba1)(9/17)<br>
-
=====① ～⑥ colony PCR lacP+kil+DT=====
+
lane3, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3)<br>
-
 
+
lane4, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl, Spel)<br>
-
次の写真
+
lane5, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl)<br>
-
=====① 　　　1000kb ladder =====
+
lane6, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (Spel)<br>
-
=====② 　　　Positive control (GFP+DT)=====
+
====Liquid culture====  
-
=====③ 　　　Negative control (MilliQ)=====
+
*LacP-torA-GFP-DT(pSB4K5) (1-4)
-
=====④ ～⑨　lacP+kil+DT　①～⑥=====
+
====Restriction enzyme processing====
-
=====⑩～⑫　  pspA 10μL ,6×buffer 2μL=====
+
-
⑬      1000kb ladder
+
-
==August 15==
+
-
====Colony PCR====
+
-
pspA
+
{|class="wikitable"
{|class="wikitable"
-
!2×Quick Taq||pspA r-s||pspA f-p||MilliQ||Total
+
!J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3)!!EcoRl!!Spel!!buffer M!!MiliQ!!Total
|-
|-
-
|25||1||1||23||50
+
|15||1||1||3||10||30
|}
|}
-
94℃, 2min<br>
 
-
94℃, 30sec<br>
 
-
56℃, 30sec<br>
 
-
68℃, 1min<br>
 
-
→35cycles<br><br>
 
-
==August 16==
 
-
==August 17==
 
-
====Colony PCR====
 
-
pspA
 
{|class="wikitable"
{|class="wikitable"
-
!10×Buffer for KOD-Plus-Ver2||2mM dNTPs||25mM MgSO4||Primer PsPA  f||Primer PsPA  r||KOD-Plus-||MilliQ||Total
+
!J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3)!!EcoRl!!Pstl!!buffer H!!MiliQ!!Total
|-
|-
-
|5||3||3||1.5||1.5||1||35||50
+
|15||1||1||3||10||30
|}
|}
-
{|class="wikitable"
+
at 37℃ for 1 hour
-
!10×Buffer for KOD-Plus-Ver2||2mM dNTPs||25mM MgSO4||Primer PsPA  f||Primer PsPA  r||KOD-Plus-||MilliQ||Total
+
-
|-
+
-
|5||3||5||1.5||1.5||1||33||50
+
-
|}
+
-
negative control(MilliQ 50μL)
+
-
 
+
-
94℃, 2min<br>
+
-
94℃, 15sec<br>
+
-
55℃, 30sec<br>
+
-
68℃, 1min<br>
+
-
→35cycles<br><br>
+
====Electrophoresis====
====Electrophoresis====
-
写真貼ってください
+
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl, Spel)<br>
-
=====① 　　　1kb ladder=====
+
J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (EcoRl, Pstl)<br>
-
=====②, ③　　MgSO4 3μL, pspA=====
+
→The bands didn't appear.
-
=====④, ⑤　　MgSO4 5μL, pspA=====
+
-
=====⑥      negative control(MilliQ)=====
+
-
=====⑧      1kb ladder=====
+
-
==August 18==
+
-
==August 19==
+
-
==August 20==
+
-
 
+
-
 
+
 +
 +
</div>
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Latest revision as of 18:19, 25 September 2012

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Contents

Secretion Notebook

February 7

Preculture

We started preculture at 12:10.

February 8

Culture

We start culturing with 300[mL] of LB medium.

timeOD600
12:00start
14:100.019
14:450.154
15:050.267
15:210.64

Making Competent Cell

We made competent cells.

Transformation

pGEM_TAP
LacP (BBa_R0011)
DT (BBa_B0015)

Making Culture Medium Plates

We made 200mL of ampicillin culture, kanamycin culture, and chloramphenicol culture.

Transformation

GFP(BBa_E0040) in pSB1A2
DT(BBa_B0015) in pSB1AK3
ara(BBa_I0500) in pSB2K3
LacP(BBa_R0011) in pSB1A2

February 9

transformation

BBa-E0040(GFP)(Mr.Fujita)

Liquid culture

DT.leap colony transformed on February 8
colony of competent cell made on February 8

February 10

Miniprep

DT2 43.9μg/ml(1.34 260/230 1.74 260/280)
LacP1 17.1μg/ml(1.54 260/280 0.83 260/230)
LacP2 18.0μg/ml(1.58 260/280 0.87 260/230)

transformation

B0040 1.4k PsB1A2 B0034 1.2M pSB1A2(from iGEM parts plate)

Competent cell

We did preculture for overnight. We put 1.5mL of preculture on 150mL of LB culture.

timeOD600
11:45start
13:300.048
14:300.168
15:030.256
15:200.405
15:350.459
at last0.576

February 11

Checking Transformation efficiency

Conpetent cell's transformation efficiency is 1.3x10^4colonys/μg

February 13

Transformation

Const promoter J23110, J23109, J23100

DNACompetent celltotal
1μL2021

No colony was there on February 14

Liquid culture

LacP, DT, RBS(BBa_B0034),GFP
start at 20:00
in Plus grow with Ampicilin 3mL

February 14

Miniprep

concentration[μg/μL]

LacP339.6
LacP440.8
LacP528.9
RBS128.2
RBS257.4
RBS313.2
DT369.7
DT464.4
DT561.5
GFP164.0
GFP250.5
GFP366.0

Restriction

Const promoterJ23100

DNASpe1Pst1Buffer2BSAMilliQtotal
200.50.530.55.530

at 37℃ for overnight

February 15

Making gel

1% Agarose gel

AgaroseTAE
1.6g160mL

Electrophoresis

No.1

Gel No.1

Restriction productloading dye
5μL1

The marker was 1kb ladder
It seemed that this restriction product was not cut.

No.2

Gel No.2
Lane1 : 1kb ladder
Lane2 : J23100 2μL + 6*Loading dye 1μL
Lane3 : J23100(Spe1,Pst1) 5μL
Lane4 : J23100(Spe1,Pst1) 2μL

  • There were bands on lane_2 and we cannot identify these bands because the sample of lane_2 was not cut with any restriction enzyme.
  • There must have been bands at 2100bp and 883bp on lane_3 and lane_4.

Testing whether restriction enzyme were deactivated or not

DNA(DT)restriction enzymeBufferBSAMilliQtotal
100.530.51630

at 37℃ for Oveernight
Restriction enzyme means Spe1(1,2) Pst1(1,2,3) in this time.











February 16

Electrophoresis

Electrophoresis021601.JPG

1. 1kb ladder
2. DT2
3. DT3
4. DT2 (Spe1-1)
5. DT2 (Spe1-2)
6. DT2 (Pst1-1)
7. DT2 (Pst1-2)
8. DT2 (Pst1-3)
9. DT3 (Pst1-4)
10. 1kb ladder

Pst1-1, Pst1-2, and Pst1-3 did not cut DNAs. They seemed to be deactivated.

Genomic PCR

10*Buffer for KOD Plus2mM dNTPs25mM MgSO410μM primer-f10μM primer-r158ng/μL Genomic DNAKOD plusMilliQtotal
5531.51.5113250

Electrophoresis

Electrophoresis021602.JPG

1. 1kb ladder
2. tatABCD (2.5kb)
3. TAMO reductase (2.7kb)
4. Negative control
We got bands of tatABCD but there were nonspecific amplification products.
We failed amplification of TAMO reductase.







Transformation

Constitutive promoter (BBa_J23107 , BBA_J23117)
High copy plasmid (pSB1AT3)

DNAcompetent cell
1μL10μL

February 17

PCR

We did PCR to amplify products of PCR that we had done yesterday but we could not amplify tatABCD.

Genomic PCR

BufferdNTPsMgSO4primer-fprimer-rgenomic DNAKOD plusMilliQtotal
2.52.550.750.750.50.51650

Predenature 94℃, 2min
Denature 98℃, 10sec
Annealing 57℃, 30sec
Extension 68℃, 2.5min
(30cycles)

Electrophoresis

Electrophoresis021701.JPG

1. 1kb ladder
2. TAMO reductase
3. Negative control

Restriction

J23100Spe1Pst1Buffer2BSAMilliQtotal
100.50.530.515.530

Genomic PCR

BufferdNTPsMgSO4primer-fprimer-rgenomic DNAKOD plusMilliQtotal
TMAO reductase2.52.530.750.750.50.515.525
tatABCD2.52.520.750.750.50.515.525
tatABCD2.52.520.750.7550.510.525

Electrophoresis

Electrophoresis021702.JPG

1. 1kb ladder
2.3. TAMO reductase
4. tatABCD
5. tatABCD(10 times amount of genome)

Checking of restriction enzyme

DTenzymeBufferBSAMilliQtotal
20.530.52430

at 37℃ for overnight
We checked EcoR1 and Xba1.







February 18

Miniprep

μg/mL260/280230/260
JS3117-11351.52.06
JS3117-2751.61.63
JS3109-11151.51.88
JS3109-2751.651.71
pSB1AT3-1701.661.83
pSB1AT3-21001.521.54

diluted to 25 times

Competent cell

We put 3mL of preculture product on yesterday onto 300mL of LB medium

timeOD600
10:30start
12:100.118
13:000.270
13:300.502

Transformation

pSB1AT3-2competent cellMilliQtotal
0201030
220830
1020030
  • Results(on Feb. 19)
pSB1AT3-2number of colony
00
2177
10590

Transformation efficiency 7.4x10^4 colonys/μg

February 20

Restriction

sample 1

DT plasmidEcoR1Xba1BufferBSAMilliQtotal
7.50.50.530.51830

sample2

GFP plasmidEcoR1Spe1BufferBSAMilliQtotal
100.50.530.515.530

Electrophoresis

  • sample1
Electrophoresis022001 .JPG

a. sample1 2μL + MilliQ 3μL + 6×Loading Dye 1μL
b. sample1 5μL + 6×Loading Dye 1μL
lane_1 1kb ladder
lane_2 a
lane_3 b
lane_4 a
lane_5 b
lane_6 a
lane_7 b
lane_8 1kb ladder

  • sample2
Electrophoresis022302.JPG

c. sample2 2μL + MilliQ 3μL + 6×Loading Dye 1μL
d. sample2 5μL + 6×Loading Dye 1μL
lane_1 1kb ladder
lane_2 c
lane_3 d
lane_4 1kb ladder

PCR

BufferdNTPsMgSO4primer-fprimer-rgenomic DNAKOD plusMilliQtotal
tatABCD12.52.51.50.750.750.50.51625
tatABCD22.52.51.50.750.750.50.51625
TMAO reductase12.52.520.750.750.50.515.525
TMAO reductase22.52.520.750.7550.510.525

Predenature 94℃ 2min
Denature 98℃ 10sec
Annealing 59℃ 30sec
Extension 68℃ 2.5min
→30cycles

BufferdNTPsMgSO4primer-fprimer-rPCR productsgenomic DNAKOD plusMilliQtotal
tatABCD12.52.51.50.750.7500.50.51625
tatABCD22.52.51.50.750.75100.515.525
TMAO reductase12.52.520.750.75000.51625
TMAO reductase22.52.520.750.75000.51625

Predenature 94℃ 2min
Denature 98℃ 10sec
Annealing 59℃ 30sec
Extension 68℃ 2.5min
→35cycles

Electrophoresis

Electrophoresis022003.JPG

lane_1.1kb ladder
lane_2.tatABCD
lane_3.tatABCD
lane_4.TAMO1
lane_5.TAMO2
lane_6.1kb ladder




Electrophoresis022004.JPG

lane_1.1kb ladder
lane_2.tatABCD1
lane_3.tatABCD2
lane_4.TAMO
lane_5.TAMO
lane_6.1kb ladder





February 21

PCR (Advantage HF protocol)

bufferdNTPsprimer-fprimer-rgDNAPCR productsDWpolymerasetotal
tatABCD2.52.50.750.7510170.525
TMAO2.52.50.750.7501170.525

Predenature 94℃ 1min
Denature 94℃ 30sec
Annealing 58℃ 30sec
Extension 68℃ 3min
→25cycles

Electrophoresis

Electrophoresis0221.JPG

1. 1kb ladder 2μL
2. tatABCD 5μL + 6×Loading Buffer 1μL
3. TAMO 5μL + 6×Loading Buffer 1μL
4. 1kb ladder 2μL

Liquid culture
pSB3C5-1,2
pSB4K5-1,2

February 22

Gel extraction

lane 1 of the gel 45.0μg/mL

PCR purification

product 38.2μg/mL

PCR

TMAO reductase

BufferdNTPsMgSO4prefix primer-fsuffix primer-rproduct of gel extractproduct of PCR purification(1ng/μL)KOD plusMilliQtotal
15541.51.50.50132.550
25541.51.510132.550
35541.51.500.5132.550
45541.51.501132.550

94℃, 2min
98℃, 10sec
59℃, 30sec
68℃, 3min
→25cycles

Electrophoresis

Electrophoresis022201.JPG

1. 1kb ladder
2. TAMO1
3. TAMO2
4. TAMO3
5. TAMO4
6. constructive promoter 1-18C
7. constructive promoter Spe1
8. constructive promoter Pst1
9. 1kb ladder

PCR

BufferdNTPsMgSO4prefix primer-fsuffix primer-rproduct of PCR purification(1ng/μL)KOD plusMilliQtotal
15531.51.50.5132.550
25531.51.5113250
35531.51.5213150
45531.51.5313050
55531.51.51012950
65531.51.5013350

94℃, 2min
98℃, 10sec
59℃, 30sec
68℃, 3min
→25cycles

Electrophoresis

Electrophoresis022202.JPG












Checking Dpn1

BufferLacP(28.7ng/μL)Dpn1MilliQtotal
2100.57.520
2100517

February 23

Colony PCR

tatABCD(2 samples)

BufferdNTPsMgSO4primer-fprimer-rKOD plusMilliQtotal
5531.51.513350

Predenature 94℃ 1min
Denature 98℃ 10sec
Annealing 59℃ 30sec
Extension 68℃ 3min
→25cycles

Electrophoresis

Electrophoresis022301.JPG

1. 1kb ladder 2μL
2. tatABCD 1 5μL + 6×Loading Buffer 1μL
3. tatABCD 2 5μL + 6×Loading Buffer 1μL
4. 1kb ladder 2μL

Miniprep

pSB4K5 and pSB3C5
deluted it to 25 times and then measured it
pSB4K5 1 : 60.0 μg/ml 1.67(260/280) 1.98(260/230)
pSB4K5 2 : 55.0 μg/ml 1.49(260/280) 1.62(260/230)
pSB3C5-3 : 3.6 μg/ml 1.57(260/280) 3.00(260/230)
pSB3C5-4 : 1.3 μg/ml 1.44(260/280) 1.04(260/230)

Liquid culture

pSB3C5-3,4

Electrophoresis

Electrophoresis022302.JPG

1. 1kb ladder 2μL
2. pSB3C5-3 5μL, 6×loading dye 1μL
3. pSB3C5-4 5μL, 6×loading dye 1μL
4. 1kb ladder 2μL







February 27

Test of Dpn1

Buffer2GFP2BSAMilliQDpn1
330.3231

Colony PCR

bufferdNTPsMgSO4Primer-fPrimer-rMilliQKOD Plustotal
Colony PCR(2 samples)5531.51.533150
Negative control5531.51.534050

Predenature 94℃,2min
Denature 98℃,10sec
Annealing 59℃,30sec
Extension 68℃,3min
→25cycles

Electrophoresis

Electrophoresis0227.JPG
sampleLoading DyeMilliQ
1.1kb ladder200
2.product of PCR1510
3.product of PCR2510
4.product of PCR(Negative control)510
5.product of PCR(2/23)510
6.GFP2(DPN1)1020
7.GFP2327
8.1kb ladder200

Results of liquid culture

We measure this after dilute it to 10 times.

pSB3C5-5pSB3C5-6pSB3C5-5(1% glucose)pSB3C5-6(1% glucose)
8.5[µg/ml]-1.8-17.9-18.2

PCR

bufferdNTPsMgSO4Primer-f(prefix)Primer-r(suffix)PCR purification product(1ng/µL)MilliQKOD Plustotal
15531.51.50.232.8150
25531.51.50.532.5150
  • PCR purification product was that purification product(75ng/µL) of electrophoresis-3 deluted to 1ng/µL

Predenature 94℃,2min
Denature 98℃,10sec
Annealing 59℃,30sec
Extension 68℃,3min
→25cycles

February 28

Electrophoresis

Electrophoresis022801.JPG

1. 1kb ladder
2. PCR1 →Product of gel extraction : tatABCD with prefix and suffix 105[ng/µL]
3. PCR2

Restriction

Buffer2plasmid(?)enzymeMilliQtotal
220.215.820

incubate 1 hour at 37℃

Electrophoresis

Electrophoresis022802.JPG
Electrophoresis022803.JPG

1. 1kb ladder
2. Control (without enzymes)
3. EcoR1
4. Xba1 (crystallized)
5. Xba1 (with seal)
6. Spe1
7. Pst1
8. 1kb ladder

PCR and Electrophoresis

Quick Taq Dye Mixprimer-fprimer-rtemplateMilliQtotal
251.01.00.522.550

Predenature 94℃,2min
Denature 94℃,30sec
Annealing 59℃,30sec
Extension 68℃,3min
→25cycles

Restriction

BufferHtatABCDEcoR1Spe1MilliQtotal
250.20.212.620

PCR purification

We eluted the product for 30µL MilliQ

Ligation

Insert(tatABCD)Vector(pSB1C3)Ligation Hightotal
101516

4℃, overnight

February 29

Transformation

tatABCDcompetent celltotal
11011

Checking Restriction enzyme

plasmid seems to be 1-18C promoterEnzymeBufferMilliQtotal
20.2215.820

Checking tatABCD

tatABCDHind3BufferMilliQtotal
50.2212.820

PCR

bufferdNTPsMgSO4Primer-fPrimer-rColE1(6.5ng/µL) / TMAOMilliQKOD Plus Neototal
Kil2.52.51.50.750.750.5160.525
TMAO2.52.51.50.750.750.5160.525

Predenature 94℃,2min
Denature 98℃,10sec
Annealing 60℃,30sec
Extension 68℃,3min
→30cycles

Electrophoresis

  • evernoteに写真なし！至急求む

1. 1kb ladder
2. Kil (649bp)
3. TMAO (2720bp)
4. TMAO (Quick Taq)
5. tatABCD (Quick Taq)
6. tatABCD (Hind3)
7. 1kb ladder

March 1

PCR

  • TMAO

Template is gDNA and product of colony PCR gel extraction

BuffergNTPsMgSO4Primer-fPrimer-rKOD Plus NeoTemplate gDNAproduct of gel extractionDWtotal
12.52.51.50.750.750.50.501625
22.52.51.50.750.750.50214.525

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 1.5min
→25cycles

  • Kil
BufferdNTPsMgSO4primer-fprimer-rcolE1(6.5ng/µL)KOD Plus NeoMilliQtotal
2.52.51.50.750.750.5160.525

94℃, 2min
98℃, 10sec
61℃, 30sec
68℃, 1min
→20cycles

Electrophoresis

Electrophoresis030101.JPG
Electrophoresis030102.JPG

1. 1kb ladder
2. TMAO1 (gDNA)
3. TMAO2 (product of gel extraction)
4. Kil

PCR

BufferdNTPsMgSO4primer-fprimer-rProduct of PurificationKOD Plus NeoMilliQtotal
5531.51.5132150

94℃, 2min
98℃, 10sec
61℃, 30sec
68℃, 30sec
20cycles
→Purification 230ng/µL

Restriction

KilEcoR1Spe1BufferHMilliQtotal
50.20.2212.620

incubate at 37℃, for 1.5 hours

PCR Purification

Ligation

KilpSB1C3Ligation Hightotal
5139

at 4℃, for overnight

March 2

Ligation

KilpSB1C3Ligation High Ver.2total
5139
tatABCDpSB1C3Ligationtotal
101516

at 16℃ for 1 hour

Transformation

KilKil(3/1,Ligation)tatABCDcompetet celltotal
1001011
0101011
0011011

PCR

Quick Taqprimer-rprimer-ftemplateMilliQtotal
25110.522.550

Electrophoresis

Electrophoresis030227.JPG



Restriction

pSB3C5-5EcoR1Pst1BufferHBSAMilliQtotal
200.50.530.55.530

at 37℃, for 2 hour

Electrophoresis

Electrophoresis030225.JPG

1. 1kb ladder 2µL
2. pSB3C5 5µL + 6×Loading Buffer 1µL
・product of gel extraction(about 2700bp)
－30.9µg/mL

Restriction

GFP1 ,2 ,3

GFPEcoR1Pst1BufferBSAMilliQtotal
100.50.530.515.530

at 37℃, for 2.5 hours

Restriction

DTEcoR1Xba1BufferMBSAMilliQtotal
100.20.230.316.330
Constitutive PromoterSpe1Pst1BufferMBSAMilliQtotal
150.20.230.311.330

at 37℃, 2 hours

  • J23117-1:135ng/µL, J23107-1:115ng/µL
  • DT3→PCR Purification
  • Promoter→Gel Extraction

Checking TMAO

something seems to be TMAOBuffer2EcoR1MilliQtotal 1020.57.520

at 37℃, for 1 hour

Electrophoresis

Electrophoresis030226.JPG

1. 1kb ladder
2. GFP1 that had been cut by restriction enzyme
3. GFP2 that had been cut by restriction enzyme
4. GFP3 that had been cut by restriction enzyme
5. GFP1
6. GFP2
7. GFP3
8. TMAO (control)
9. TMAO (EcoR1)
10. DT (control)
11. DT (EcoR1, Xba1)
12. 1kb ladder

Checking tatABCD

Quick Taqprimer-fprimer-rMilliQtotal
25112350

Electrophoresis

Electrophoresis030228.JPG













March 3

PCR

templatebufferdNTPsMgSO4VFVRKOD plusMilliQtotal
115531.51.513250
225531.51.513150

94℃, 2min
98℃, 10sec
50℃, 30sec
68℃, 1min
→30cycles

Miniprep

March 4

Sequence of tatABCD

Quick Taqprimer-fpromer-r sequencetemplateMilliQtotal
251112350
Quick Taqprimer-f sequenceprimer-rtemplateMilliQtotal
251112350

Colony PCR of TMAO

bufferdNTPsNgSO4primer-fprimerr-rKOD plusMilliQtotal
5541.51.513250

→ethanol precipitation
94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 2.5min
→25cycles

Electrophoresis

1. 1kb ladder
2. tatABCD1
3. tatABCD2
4. TMAO
5. 1kb ladder

Restriction

TMAOEcoR1BufferHBSAMilliQtotal
100.230.316.530
TMAOXba1Pst1BudderMBSAMilliQtotal
100.20.230.316.330

at 37℃ for 1 hour

Electrophoresis


Transformation

pSB1C3competent cell(made at 2/8)total
5100105

March 5

Restriction

pSB1C3(Xba1, Spe1)Pst1BufferHBSAMilliQtotal
100.220.27.620
pSB1C3(Xba1, Spe1)EcoR1BufferHBSAMilliQtotal
100.220.27.620

at 37℃ for 1 hour
→Then we did ethanol precipitation

Ligation

Kil(EcoR1, Spe1)pSB1C3(EcoR1)Ligation Hightotal
5139

at 16℃ for 1 hour

Transformation

Kilcompetent celltotal
11011

We used commercially available competent cells in this time.

PCR

TMAO

bufferdNTPsMgSO4Primer-fPrimer-rTemplateKOD plusMilliQtotal
5531.51.5113250

Electrophoresis

(31)

Restriction

LacPpSB3C5EcoR1Pst1BufferHBSAMilliQtotal
12000.50.530.55.530
20200.50.530.55.530

at 37℃ for 1 hour
1→Ethanol precipitation　45.4µg/mL
2→Gel extraction　38.7µg/mL

Ligation

LacPpSB3C5Ligation Hightotal
102618

at 4℃ for overnight

Transformation

LacP+pSB3C5competent celltotal
11011

on ice for 30 mins.
heat shock at 42℃ for 60secs
on ice for 2 mins.
After we incubate with 200µL of SOC culture for 1 hour, we did plating on LB culture with CP

Restriction

GFP PlasmidEcoR1Spe1Buffer2BSAMilliQtotal
100.50.530.515.530

at 37℃ for 2 hours

Electrophoresis

1. Ladder 2µL
2. GFP Plasmid (already restricted) 2µL + Loading Dye 2µL + MilliQ 8µL
3. Ladder 2µL

PCR

torA signal and pspA pspAはコロニーPCR

BufferdNTPsMgSO4Primer-fPrimer-rtemplate(TMAO)KOD plusMilliQtotal
2.52.51.50.750.750.50.51625

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec

electrophoresis

1. 100bp Ladder
2. torA signal (272bp)
3. pspA (969bp)
4. 100bp Ladder

March 6

Restriction

GFPEcoR1Spe1BufferMBSAMilliQtotal
120.50.530.513.530

We did incubate at 37℃ for 1.5hours.
And we did gel extraction on 3/7 and get 40.0μg/mL GFP.

PCR

bufferdNTPsMgSO4Primer-fPrimer-rtemplateKOD plus neoMilliQtotal
5531.51.50.5132.550

94℃　2min
98℃　10sec
60℃　30sec
68℃　(torA 10sec / pspA 30sec) 25 cycles
We used product of PCR on 3/5 of torA and pspA as template.

Restriction

KilEcoR1Spe1BufferMBSAMilliQtotal
100.30.330.316.130
DTEcoR1Xba1Buffer2BSAMilliQtotal
100.50.530.515.530

at 37℃ for 2 hours
→ purification 37.7ng/μL

Ligation

KilpSB1C3Ligation High Ver.2total
4239
  • Kil : 350fmol
  • pSB1C3 : 29fmol

at 16℃ for overnight

March 7

Electrophoresis

Electrophoresis0307.JPG

1. 1kb ladder 2µL
2. pspA (PCR product)2.5µL + Loading Dye 0.5µL
3. GFP 30µL + Loading Dye 6µL
4. 1kb ladder 2µL

  • The GFP was gel extracted on 3/6 and concentrated by Vacuum in 150µg/mL

Ligation

VectorDNAGFPLigation High Ver.2total
5151030

Restriction

torAEcoR1Spe1bufferMBSAMilliQtotal
100.30.330.316.130
pspAEcoR1Spe1bufferMBSAMilliQtotal
50.30.330.321.130

at 37℃ for 1.5 hours

Purification

torA→31.8ng/µL
pspA→49.3ng/µL

Ligation

torApSB1C3Ligation High Ver.2total
3339
pspApSB1C3Ligation High Ver.2total
4239

at 4℃, for overnight

  • torA→31.8ng/µL×3µL=95.4ng=0.529pmol
  • pSB1C3→19.4ng/µL×3µL=58.2ng=0.042pmol
  • pspA→49.3ng/µL×4µL=197.2ng=0.308pmol
  • pSB1C3→19.4ng/µL×2µL=38.8ng=0.029pmol

March 8

Restriction

pSB4K5EcoR1Spe1BufferMBSAMilliQtotal
200.20.230.26.430

at 37℃ for 1 hour.
→Purification : 36.6ng/µL

Ligation

KilpSB4K5Ligation High Ver.2total
101516

at 4℃ for overnight

  • Kil→37.7ng/µL×10µL=377ng=879fmol
  • pSB4K5→36.6ng/µL×1µL=36.6ng=86fmol

Liquid culture

LacP + pSB3C5 -1, 2

Transformation

torApspAcompetent celltotal
101011
011011

We use commercially available competent cells in this time.

March 9

Restriction

tatABCDXba1Pst1BufferMBSAMilliQtotal 100.20.230.316.330

at 37℃ for 1hour
→purification : 75.0μg/mL (1.11 260/280 , 0.81 260/230)

Miniprep

LacP + pSB3C5 1 80.5μg/mL (1.78 260/280 , 2.00 260/230)
LacP + pSB3C5 2 107.2μg/mL (1.83 260/280 , 1.90 260/230)

Colony PCR

Quick TaqVFVRMilliQtotal
25112350

94℃ 2min
94℃ 30sec
55℃ 30sec
68℃ 6sec
25cycles

Ligation

tatABCDconstP J23107Ligation High Ver.2total
5139

tatABCD : 227fmol
constP J23107 : 21fmol

Transformation

pspAtorAKilcompetent cell
110010
201010
300110

Miniprep

4mL of plusgrow which had been cultured for overnight.
pSB4K5 : 80.5μg/mL

March 10

Screening PCR

Quick TaqVFVRMilliQtotal
25112350
Electrophoresis0310.JPG

Then we did electrophoresis to confirm.
1.1kb ladder
2.kil (649bp)
3,4,5, pspA (969bp)
6,1kb ladder

1, 100bp ladder
2,3,4, torA signal

Restriction

LacP-pSB3C5Spe1Pst1BufferMBSAMilliQtotal
100.20.220.27.420

for 2.5 hours at 37℃
→purification 29.0ng/μL

torAXba1Pst1BufferMBSAMilliQtotal
100.20.220.27.420

for 2.5 hours at 37℃
→purification 91.8ng/μL

Ligation

torALacP-pSB3C5Ligation High Ver.2total
4239

torA : 929fmol
LacP-pSB3C5 : 284fmol
for overnight at 4℃

March 11

Miniprep

We used 3μL of plus grow that we had cultured for overnight.
torA : 62.8ng/μL

Screening PCR

Quick TaqVFVRMilliQtotal
25112350

Electrophoresis

Electrophoresis031101.JPG

1. 1kb ladder
2〜11. pspA (969bp)
12. 1kb ladder

The results were shown as photograph in the right.

It seemed that there were shorter sample than expected sample, so we did electrophoresis with pspA which was product of PCR and pspA which had already cut with EcoR1 and Spe1.





Electrophoresis031102.JPG

1.1kb ladder
2. pspA (PCR product)
3, pspA (Eco, Spe)
4〜6, pspA (colony PCR)
7,1kb ladder

The results were shown as photograph in the right.

March 12

Transformation

DT(1ng/μL)DT(0.1ng/μL)KilLacP-torAMilliQcompetent celltotal
100002021
010002021
005005051
000505051
000012021

Restriction

pspAEcoR1Spe1BufferMBSAMilliQtotal
100.50.530.315.730

at 37℃ for 4 hours
→ We did purification and got 48.3ng/μL pspA.

Ligation

pspApSB1C3MilliQLigation High Ver.2total
42039
22026
02226

March 13

Miniprep

pSB1C3 74.2µg/mL 1.65 (260/280) 1.31 (260/230)

March 14

Restriction

pSB1C3EcoR1Spe1BufferMBSAMilliQtotal
200.20.240.415.240

We did gel extraction and got 47.2ng/µL pSB1C3 but we did not cut out RFP.

Ligation

torApSB1C3Ligation High Ver.2total
4239
MilliQpSB1C3Ligation High Ver.2total
4239

at 16℃, for 1 hour

  • torA : 0.707pmol
  • pSB1C3 : 0.068pmol

Liquid culture

We cultured LacP-pSB3C5 1,2,3,4,5 (CP tolerance)on culture with Amp.
→Only 4 which did not be cultured succeeded.

March 15

Liquid culture

We cultured LacP-pSB3C5 6,7,8,9,10 on culture with ampicillin.
→6,8,9,10 were succeeded.

Restriction

GFPEcoR1Spe1Buffer2BSAMilliQtotal
100.50.530.515.530
DTEcoR1Xba1Buffer2BSAMilliQtotal
100.50.530.515.530

for 2 hours at 37℃.

Miniprep

pspA (pSB1C3) 40.5ng/µL

Ligation

pspADTLigation High Ver.2total
51.539.5
  • pspA : 385fmol
  • DT : 36fmol

Transformation

J23107-tatABCDDT (0.1ng/µL)DT (0.01ng/µL)pspA-DTcompetent cells on 3/15total
20002022
02002022
00202022
00022022

Screening PCR

Kil, pspA and torA

Quick TaqPrimer-rPrimer-fMilliQtotal 25112350

March 16

Miniprep

torA (pSB1C3) 68.8ng/µL
Kil (pSB4K5) 92.7ng/µL
torA was red for some reason. We do not know why.

Colony PCR

Quick TaqPrimer-rPrimer-fMilliQtotal
25112350

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 6sec
→25cycles

Electrophoresis

Electrophoresis0316.JPG

The results were shown as photograph in the right.

Checking Transformation Efficiency

competent cells that were made on March 15.
DNA : 0.02ng → 668 colonies Transformation Efficiency : 3.3×10^7
DNA : 0.2ng → 1739 colonies Transformation Efficiency : 8.7×10^6

Restriction

pSB1C3EcoR1Spe1BSABufferMBufferHMilliQtotal
200.20.20.3306.330
50.200.20212.620

at 37℃, for 2 hours.
We did gel extraction for product with EcoR1, Spe1. We got 46.1ng/µL pSB1C3.

Kil(pSB4K5)EcoR1Pst1BSABufferHMilliQtotal
50.20.20.2212.420
50.200.2212.620

for overnight at 37℃.
We did this to confirm.

pspA (pSB1C3)EcoR1Pst1BSABufferHMilliQtotal
50.20.20.2212.420
50.200.2212.620

for overnight at 37℃.
We did this to confirm.

Ligation

torApSB1C3Ligation High Ver.2total
4239
MilliQpSB1C3Ligation High Ver.2total
4239

at 16℃, for overnight

  • torA→767fmol
  • pSB1C3→68fmol

March 17

Miniprep

J23107-tatABCD 72.7ng/µL
pspA-DT 50.5ng/µL

Checking the Insert

J21037-tatABCDEcoR1Pst1BSABufferHMilliQtotal
50.20.20.2212.420
50.200.2212.620

Success.

pspA-DTEcoR1Pst1BSABufferHMilliQtotal
50.20.20.2212.420
50.200.2212.620

Failed.

March 19

Restriction

DTEcoR1Xba1BSABufferMMilliQtotal
100.20.20.3316.330

We did Gel extraction and got 17.2ng/µL of DT.

Ligation

KilDTLigation High Ver.2total
102618

We did this for an hour at 16℃.

Restriction

GFPEcoR1Spe1BSABufferMMilliQtotal
100.50.50.5315.530

We did this for 4 hours at 37℃

Ligation

pspADTLigation High Ver.2total
55515
pspApSB1C3Ligation High Ver.2total
4138

We did these for an hour at 16℃.

  • pspA (5µL)→377fmol
  • DT→39fmol
  • pspA (4µL)→339fmol
  • pSB1C3→34fmol

Transformation

pspA-DT, pspA (pSB1C3), torA (pSB1C3) and GFP-DT

March 20

Screaning PCR

Quick TaqPrimer-RPrimer-FMilliQtotal
25112350
  • pspA→○
  • pspA-DT→○
  • GFP-DT→○
  • torA→×
  • Kil-DT 6 of 8 sumples→○
Quick TaqVRVFMilliQtotal
25112350
  • pspA→○
  • pspA-DT→×

Restriction

torAEcoR1Spe1BSABufferMMilliQtotal
100.30.30.3316.130
DTEcoR1Xba1BSABufferMMilliQtotal
100.20.20.3316.330

We did this for overnight at 37℃. And we did purification.
torA 34.2ng/µL
DT 28.0ng/µL

March 21

Miniprep

GFP-DT-1 : 77.7ng/µL
GFP-DT-2 : 67.6ng/µL

Restriction

pSB1C3EcoR1Spe1BSABufferMMilliQtotal
100.20.20.3316.330
50.200.2212.620

We did this for 3 hours at 37℃, and then we did gel extraction. We got 25.1ng/µL pSB1C3

GFP-DTEcoR1Pst1Xba1BSABufferMMilliQtotal
50.20.200.2212.420
50.2000.2212.620
100.200.20.3316.330

We did this for 3 hours at 37℃, and then we did Purification. We got 30.7ng/µL GFP-DT.

Ligation

torApSB1C3pspADTGFP-DTLigation High Ver.2total
14100038
201700412
300530412
430005412

We did this for an hour at16℃.

March 22

PCR

We did PCR to amplify torA_signal that was product of PCR at March 5 with redesigned primers.

BufferdNTPsMgSO4Primer-FPrimer-RTemplateMilliQKOD plus neototal
5531.51.50.532.5150

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 10sec
→30cycles
→Purification 110.7ng/µL

Restriction

torAEcoR1Spe1BSABufferMMilliQtotal
100.20.20.3316.330

Ligation

torApSSB1C3pspADTGFP-DTLigation hightotal
143000411
23000339
300550515
  • torA (4µL)→512fmol
  • pSB1C3→54fmol
  • torA (3µL)→384fmol
  • GFP-DT→36fmol
  • pspA→377fmol
  • DT→65fmol

March 23

Screening PCR

torA (pSB1C3), torA-GFP-DT and pspA-DT

Quick TaqVFVRMilliQtotal
25112350

E.coli in liquid culture that had pspA(pSB1C3) also expressed RFP.

Restriction

pspAEcoR1Spe1BufferMBSAMilliQtotal
50.30.330.321.130

And then we did ethanol precipitation

Ethanol precipitation

pspA 11.5ng/µL.

Miniprep

LacP+pSB3C5-8 77.9µg/mL
LacP+pSB3C5-10 69.0µg/mL
Kil+DT-4 58.0µg/mL
Kil+DT-7 15.2µg/mL

Restriction

LacP+pSB3C5-8Spe1Pst1Buffer2BSAMilliQtotal
200.50.530.55.530
Kil+DT-4Xba1Pst1Buffer2BSAMilliQtotal
200.50.530.55.530

at 37℃ for 1.5 hours
And then we did Gel extraction.

Gel Extraction

LacP+pSB3C5-8 40.4µg/mL
Kil+DT-4 26.8µg/mL

March 26

Miniprep

torA(pSB1C3) 18.5[ng/µL]
torA-GFP-DT 20.0[ng/µL]

Restriction

torA(pSB1C3)EcoR1Pst1BufferHBSAMilliQtotal
50.20.220.212.420
50.2020.212.620
torA-GFP-DTEcoR1Xba1Pst1BufferHBufferMBSAMilliQtotal
50.200.2200.212.420
50.200200.212.620
2000.20.2030.36.330

We did Gel extraction and then got ??? 28.7[ng/µL]

Ligation

LacP (pSB3C5)torA-GFP-DTLigation High Ver.2total
1539
  • LacP : 22fmol
  • torA-GFP-DT : 197fmol
pspApSB1C3Ligation High Ver.2total
101516
pspADTLigation High Ver.2total
101516
  • pspA : 180fmol
  • pSB1C3 : 18fmol
  • DT : 16fmol
LacP(pSB3C5)Kil-DTLigation High Ver.2total
1539

for 2 hours at 16℃

Transformation

LacP-Kil-DTcompetent celltotal
11011

March 27

Miniprep

We retryed miniprep of torA(pSB1C3).
We got torA and its concentration was 39.3[ng/µL].

Transformation

NameWellSampleCompetent CellsTotalPlateColony
BBa_K117004 14J(2011 plate2)520???

We added 100[µL] of culture medium before we started culturing the E.coli.

Screening PCR

Quick TaqVF2VRMilliQTotal
25112350

LacP-torA-GFP-DT 1, 3, 5, 6, 8, 9 was successful.
pspA-DT was failed.
E.coli that had pspA(pSB1C3) did not make any colony.
LacP-Kil-DT 1,2,3,4,5,6,7,8 was failed.

Liquid culture

LacP-torA-GFP-DT

July 23

Transformation

DT(64.4ng/μl)Competent cellPlating in SOC mediumTotal
120100121

July 24

Plating in SOC medium

July 25

Transformation

  • BBa_J23113 from iGEM Kit
  • LacP from iGEM Kit

July 25

July 26

July 27

Restriction Enzyme Processing

Kil +DT (44.0 ng/μl)10xBuffer2BSAXbalMilliQTotal
13.63.00.50.511.930.0
LacP(28.7ng/μl)10xBuffer2 BSASpe1Pst1Total
20.93.00.50.54.630.0

July 30

Ligation

kil+DT(Xba1,Pst1)LacP(Spe1,Pst1)Ligation High Total
3063672

July 31

Restriction Enzyme Processing

torA-GFP+DT10×M BufferBSAMilliQXba1Pst1Total
2.53.00.523.40.30.330.0

Liquid Culture

J23113(backbone J61002)

August 1

Restriction Enzyme Processing

LacP+kil+DT BSA10×H BufferEcoR1Pst1MilliQTotal
5.00.53.00.50.520.530.0
37℃ 2h
LacP middle copy BSA10×H BufferEcoR1Pst1MilliQTotal
10.00.53.00.50.515.530.0
37℃ 2h

MIniprep

J23113(backbone J61002)

Liquid culture

J23113(backboneJ61002) Three test tubes of 4 mL LB medium with ampicillin 37℃ overnight

August 2

August 3

Restriction Enzyme processing

pSB4K5BSA 10×H BufferEcoR1Pst1MilliQTotal
8.00.53.00.50.517.530.0
B0034BSA10×H BufferSpe1Pst1MilliQTotal
12.00.53.00.50.513.530.0

37℃ 2h

DNA purification

Ligation

LacP+kil+DTpSB4K5ligation highTotal
15151545
16℃ 1h

Miniprep

  • J23113(backbone J61002) 218.0μg/mL
  • J23113(backbone J61002) 252.5μg/mL
  • J23113(backbone J61002) 201.0μg/mL
  • pSB3C5 45.0μg/ml 1.60 260/280 1.80 260/230
  • pSB4C5 213.0μg/mL 1.77 260/280 4.40 260/230

August 4

August 5

August 6

August 7

August 8

August 9

August 10

Ligation

B0034 Spe1 Pst1GFP+DT Xba1 Pst1ligation highTotal
2349
16℃ 1h

Transformation

  • RBS+GFP+DT
  • LacP+kil+DT
  • RBS（for control）
To put 2ng DNA in 20μL competent cell and leave it on ice for 30min
Heat shock for 60s at 42℃
To leave on ice for 2min
To spread on ampicillin LB plate

August 11

August 12

August 13

Liquid culture

B0034 3mL ×3 　　　

37℃　overnight

August 14

Miniprep

B0034 0μg/mL
B0034 235.5μg/mL
B0034 76.5μg/mL

Colony PCR

LacP+kil+DT ×5

2×Quick TaqVFVFMilliQTotal
25112350

25cycles
pspA

10×Buffer for KOD-Plus-Ver22mM dNTPs25mM MgSO4Primer PsPA f-pPrimer PsPA r-sKOD-Plus-MilliQTotal
5531.51.513350

Electrophoresis

0814.jpg
positive control(GFP+DT)
negative control(MilliQ)
① ～⑥ colony PCR LacP+kil+DT

次の写真

① 　　　1kb ladder
② 　　　Positive control (GFP+DT)
③ 　　　Negative control (MilliQ)
④ ～⑨　LacP+kil+DT　①～⑥
⑩～⑫　 pspA 10μL ,6×buffer 2μL
⑬　　　　1kb ladder

August 15

Colony PCR

pspA

2×Quick TaqpspA r-spspA f-pMilliQTotal
25112350

94℃, 2min
94℃, 30sec
56℃, 30sec
68℃, 1min
→35cycles

August 16

August 17

Colony PCR

pspA

10×Buffer for KOD-Plus-Ver22mM dNTPs25mM MgSO4Primer PsPA fPrimer PsPA rKOD-Plus-MilliQTotal
5331.51.513550
5351.51.513350

negative control(MilliQ 50μL) 94℃, 2min
94℃, 15sec
55℃, 30sec
68℃, 1min
→35cycles

Electrophoresis

0820.JPG
①1kb ladder
②, ③　　MgSO4 3μL, pspA
④, ⑤　　MgSO4 5μL, pspA
⑥ negative control(MilliQ)
⑧ 1kb ladder






August 18

August 19

August 20

Restriction

J23113(201.0ng/μL)Xba1Pst1BufferMBSAMilliQtotal
101130.514.530

August 21

Colony PCR

pspA

10×Buffer2mM dNTPs25mM MgSO4Primer PsPA fPrimer PsPA rKOD Plus NeoMilliQTotal
5351.51.513350
5371.51.513150
53101.51.512850

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→25cycles

Restriction

J23113(218ng/μL)Spe1Pst1BufferHBSAMilliQtotal
101130.514.530
torA-GFP-DT(20ng/μL)Xba1Pst1BufferMBSAMilliQtotal
50.60.630.520.330

Electrophoresis

0821e.jpg
① 　　　1kb ladder
②, ③　　torA-GFP-DT(Xba1,Pst1)
④, ⑤　　J23113(Spe1,Pst1)

Transformation

  • LacP-torA-GFP-DT(Backbone pSB3C5)
  • BBa_K11704(Backbone pSB1A2)

Colony PCR

0821r.jpg
  • pspA
10×Buffer2mM dNTPs25mM MgSO4Primer PsPA fPrimer PsPA rKOD Plus NeoMilliQTotal
5531.51.513350
10×Buffer2mM dNTPs25mM MgSO4Primer PsPA f-pPrimer PsPA r-sKOD Plus NeoMilliQTotal
5531.51.513350
  • negative control for pspA

We did PCR without a template.

  • GFP-DT
10×Buffer2mM dNTPs25mM MgSO4VFVRKOD Plus NeoMilliQTotal
5531.51.513350

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec

Transformation

  • LacP-torA-GFP-DT(Backbone pSB3C5)
  • J23107-tatABCD

PCR

kil, torA-GFP

10×Buffer2mM dNTPs25mM MgSO4VFVRKOD Plus NeoMilliQDNATotal
5531.51.5132150

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→25cycles

August 22

Restriction

pSB3C5EcoR1Pst1BufferHBSAMilliQtotal
13.30.50.530.512.230

Gel extraction

pSB3C5(EcoR1, Pst1)
27.2μg/mL

Transformation(the second time)

  • LacP-torA-GFP-DT(Backbone pSB3C5)
  • J23107-tatABCD

Electrophoresis

写真お願いします。
See "August 21 -Colony PCR-"
1. 1kb ladder
2. pspA(Primer pspA f&r)
3. negative control for 2
4. GFP-DT
5. pspA(Primer pspA f-p&r-s)
6. pspA(Primer pspA f-p&r-s)
7. negative control for 5&6

PCR

LacP-GFP-DT(a kit of biological parts)

10×Buffer2mM dNTPs25mM MgSO4VFVRKOD Plus NeoMilliQDNATotal
5531.51.5132150

Colony PCR

pspA

10×Buffer2mM dNTPs25mM MgSO4Primer pspA fPrimer pspA rKOD Plus NeoMilliQTotal
5531.51.513350
10×Buffer2mM dNTPs25mM MgSO4Primer pspA f-pPrimer pspA r-sKOD Plus NeoMilliQTotal
5531.51.513350

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→30cycles

Electrophoresis

写真お願いします。
1. 1kb ladder
2. pspA(Primer pspA f&r)
3. pspA(Primer pspA f-p&r-s)
4. negative control for pspA(Primer pspA f&r)
5. negative control for pspA(Primer pspA f-p&r-s)

Transformation

Kil

August 23

Colony PCR

J23107-tatABCD
VF and VR were used for amplifying approximately 2800bp

10×Buffer2mM dNTPs25mM MgSO4VFVRKOD Plus NeoMilliQDNATotal
5531.51.5132.50.550

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 90sec
→25cycles

August 24

Purification of PCR products

pspA(Primer pspA f-p&r-s)
84.1μg/mL
→See "August 22 -Colony PCR-"

Restriction

pspAEcoR1Pst1BufferHBSAMilliQtotal
200.50.530.55.530
pspAXba1Pst1BufferMBSAMilliQtotal
200.50.530.55.530

at 37℃ for 1h

Gel extraction

  • pspA(EcoR1, Pst1) 30.7μg/mL
  • pspA(Xba1, Pst1) 44.0μg/mL

Restriction

pSB1C3(82.9μg/mL)EcoR1Pst1BufferHBSAMilliQtotal
200.50.530.55.530

→Gel extraction

Electrophoresis

0824g.jpg

1. 1kb ladder
2. J23107-tatABCD →See "August 23 -PCR-"
3. negative control for J23107-tatABCD
4. pSB1C3(EcoR1, Pst1)

Ligation

pSB3C5(EcoR1, Pst1)pspA(EcoR1, Pst1)Ligation High Ver.2total
1539

16℃, overnight

August 25

August 26

August 27

Ligation

pSB1C3(EcoR1, Pst1)pspA(EcoR1, Pst1)Ligation High Ver.2total
1539

16℃, overnight

Colony PCR

Kil (1-7)

10×Buffer2mM dNTPs25mM MgSO4VFVRKOD Plus NeoMilliQTotal
5531.51.513350

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→30cycles

0827c.jpg

Purification of PCR products

torA-GFP →See "August 21 -PCR-"

Restriction

LacP(Backbone:pSB3C5)Spe1Pst1BufferHBSAMilliQtotal
200.50.530.55.530

at 37℃ for 1h
→Gel extraction

torA-GFPXba1Pst1BufferMBSAMilliQtotal
200.50.530.55.530

at 37℃ for 1h
→Purification

Purification of PCR products

J23107-tatABCD
42.0μg/mL
→See "August 23 -PCR-"

Restriction

J23107-tatABCDSpe1EcoR1BufferMBSAMilliQtotal
200.50.530.55.530

August 28

Colony PCR

Kil (1-16)

10×Buffer2mM dNTPs25mM MgSO4VFVRKOD Plus NeoMilliQTotal
5531.51.513350

File:0828c.jpg
200px

Transformation

  • pspA-pSB1C3
  • pspA-pSB3C5

Liquid culture

  • Kil-3 →Miniprep 100.4μg/mL
  • Kil-6
  • Kil-7 →Miniprep 77.7μg/mL
  • LacP-torA-GFP-1 →Miniprep 88.3μg/mL
  • LacP-torA-GFP-2 →Miniprep 85.0μg/mL
  • LacP-torA-GFP-3 →Miniprep +++

August 29

Kil assay

evernoteに結果のグラフがあります。

Liquid culture

  • pspA-pSB1C3(1-3)
  • pspA-pSB3C5(1-3)

Restriction

LacP-Kil-DT(134.7ng/μL)EcoR1Spe1BufferMMilliQtotal
101131530

37℃, overnight

Colony PCR

  • LacP-torA-GFP-DT (1-5)
  • pspA-pSB1C3 (1-5)
  • pspA-pSB3C5 (1-6)
10×Buffer2mM dNTPs25mM MgSO4VFVRKOD Plus NeoMilliQTotal
5531.51.513350

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 40sec
→30cycles

Gel extraction

J23107-tatABCD(EcoR1, Pst1) -5.9μg/mL

0829.jpg

August 30

Ligation

J23107-tatABCD(EcoR1, Spe1)22.2μg/mLDT(EcoR1, Xba1)37.5μg/mLLigation High Ver.2total
101920

at 16℃ for 1h

Transformation

J23107-tatABCD-DT (Amp resistance)

Electrophoresis

0830e.jpg

1. LacP-torA-GFP-DT-1
2. LacP-torA-GFP-DT-2
3. LacP-torA-GFP-DT-3
4. LacP-torA-GFP-DT-4
5. LacP-torA-GFP-DT-5
6. pspA-pSB1C3-1
7. pspA-pSB1C3-2
8. pspA-pSB1C3-3
9. pspA-pSB1C3-4
10.pspA-pSB1C3-5
11.pspA-pSB3C5-1
12.pspA-pSB3C5-2
13.pspA-pSB3C5-3
14.pspA-pSB3C5-4
15.pspA-pSB3C5-5
16.pspA-pSB3C5-6

Miniprep

  • pspA-pSB3C5-1 176μg/mL
  • pspA-pSB3C5-2 214μg/mL
  • pspA-pSB3C5-3 461μg/mL
  • pspA-pSB1C3-2 79μg/mL

Colony PCR

LacP-torA-GFP-DT (6-13)

2×Quick TaqVFVRMilliQTotal
25112350

94℃, 2min
94℃, 30sec
56℃, 30sec
68℃, 1min
→30cycles

Restriction

pspA-pSB1C3EcoR1Spe1BufferMBSAMilliQtotal
150.50.550.528.550

at 37℃ for 1h

Electrophoresis

0830r.jpg

1. 1kb ladder
3. pspA-pSB1C3(EcoR1, Spe1)

August 31

Gel extraction

0831e.jpg

pspA-pSB1C3(EcoR1, Spe1) 8.0μg/mL

Purification

LacP-GFP-DT -0.7μg/mL

Ligation

pspA(EcoR1, Spe1)DT(EcoR1, Xba1)Ligation High Ver.2total
2511339

at 16℃ for 1h

Transformation

pspA-DT (Amp resistance)

September 1

September 2

Colony PCR

J23107-tatABCD-DT (1-8)

2×Quick TaqVFVRMilliQTotal
25112350

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 3min
→25cycles

pspA-DT (2-8)

2×Quick TaqVFVRMilliQTotal
25112350

Extension, 10sec
→25cycles

Restriction

LacP-torA-GFP-DTEcoR1Spe1BufferHMilliQtotal
10112620

at 4℃ for overnight

Electrophoresis

0902.jpg

1. 1kb ladder
3. J23107-tatABCD-DT-1
4. J23107-tatABCD-DT-3
5. J23107-tatABCD-DT-4
6. J23107-tatABCD-DT-5
7. J23107-tatABCD-DT-6
8. J23107-tatABCD-DT-7
9. J23107-tatABCD-DT-8

0902-2.jpg

1. 1kb ladder
3. J23107-tatABCD-DT-1
4. J23107-tatABCD-DT-3
5. J23107-tatABCD-DT-4
6. J23107-tatABCD-DT-5
7. J23107-tatABCD-DT-6
8. J23107-tatABCD-DT-7
9. J23107-tatABCD-DT-8
11. LacP-pSB3C5(Spe1, Pst1)

0902-3.jpg

1. 1kb ladder
3. pspA-DT-2
4. pspA-DT-3
5. pspA-DT-4
6. pspA-DT-5
7. pspA-DT-6
8. pspA-DT-7
9. pspA-DT-8

September 3

PCR

tatABCD-DT

10×Buffer2mM dNTPs25mM MgSO4VFVRKOD Plus NeoMilliQDNATotal
5531.51.5132150

Gel extraction

0903.jpg

LacP-torA-GFP-DT →See "September 5 -Gel extraction-"

Restriction

torA-GFP-DTXba1Pst1BufferMMilliQtotal
202241240

Purification

J23107-tatABCD 29.6μg/mL 1.56(260/280) 1.60(260/230)

Electrophoresis

0903-2.jpg

1. 1kb ladder
2. J23107-tatABCD

Liquid culture

  • LacP-kil-DT(1)
  • pspA-DT(4, 7)

PCR

J23107-tatABCD

10×Buffer2mM dNTPs25mM MgSO4VFVRKOD Plus NeoMilliQDNATotal
5531.51.5129450

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 10sec
→30cycles

Gel extraction

IMG 2135.jpg

LacP-torA-GFP-DT 31μg/mL 1.28(260/280) 0.49(260/230) LacP-torA-GFP-DT 8μg/mL 1.32(260/280) 0.83(260/230)

Transformation

J23107-tatABCD (Amp resistance)
J23113-kil-DT (Amp resistance)
J23100-kil-DT (CP resistance)
J23101-kil-DT (CP resistance)
J23106-kil-DT (CP resistance)
J23115-kil-DT (CP resistance)→We didn't transform because of lack of culture medium.
J23105-kil-DT (CP resistance)→We didn't transform because of lack of culture medium.

Electrophoresis

IMG 2136.jpg

1. 1kb ladder
2-7. LacP-torA-GFP-DT

Colony PCR

LacP-torA-GFP-DT (14-21) J23113-kil-DT (Amp resistance, 1-4) J23113-kil-DT (CP resistance, 1-4)

2×Quick Taqprimer-fprimer-rMilliQTotal
25112350

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 1min
→25cycles

September 4

Liquid culture

  • LacP-kil-DT(4, 5)(Amp resistance)
  • pspA-DT(3, 6, 7)(Amp resistance)

PCR

We did PCR 10 more cycles to amplify products of PCR that we had done yesterday because we could not amplify J23107-tatABCD on September 3.
94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 1.5min
→30cycles

Electrophoresis

IMG 2137.jpg

1-8. LacP-torA-GFP-DT

IMG 2138.jpg

1-4. J23113-kil-DT (Amp resistance)
5-8. J23113-kil-DT (CP resistance)

Liquid culture

  • LacP-torA-GFP-DT
  • J23113-kil-DT
  • LacP-kil-DT(with IPTG)
  • LacP-kil-DT(without IPTG)

After 20 hour, we quantified OD600.

plasmidOD600
LacP-kil-DT(with IPTG)2.116
LacP-kil-DT(without IPTG)2.320

We cultured CP tolerance plasmid and give an antibiotic(Amp or Kan or none) after 2 hours. We quantify OD600.

time123
2hours0.1310.1000.606
add an antibioticAmpKannone
7hours0.4910.9882.634
19hours2.2530.8152.742

September 5

Colony PCR

J23107-tatABCD (1-7)

buffer2mM dNTPs25mM MgSO4primer-fprimer-rKOD Plus NeoDWTotal
5531.51.513350

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 1.25min
→30cycles

Gel extraction

by Kato
See "September 3 -Gel extraction-" LacP-torA-GFP-DT 15.9μg/mL 1.29(260/280) 0.38(260/230) LacP-torA-GFP-DT 17.2μg/mL 1.23(260/280) 0.48(260/230)

Miniprep

  • J23113-kil-DT 85.7μg/mL 1.76(260/280) 1.97(260/230)
  • LacP-torA-GFP-DT 126.6μg/mL 1.81(260/280) 2.09(260/230)
  • LacP-kil-DT-4 62.5μg/mL 1.79(260/280) 1.99(260/230)
  • LacP-kil-DT-5 56.0μg/mL 1.79(260/280) 2.01(260/230)
  • pspA-DT-3 322μg/mL 1.45(260/280) 1.42(260/230)
  • pspA-DT-6 147.6μg/mL 1.25(260/280) 1.37(260/230)
  • pspA-DT-7 145.5μg/mL 1.24(260/280) 1.27(260/230)

Restriction

pspA-DT-6Xba1Pst1BufferMBSAMilliQtotal
200.50.530.55.530

at 37℃ for 1h

Electrophoresis

IMG 2139.jpg

J23107-tatABCD (1-7)

Colony PCR

J23107-tatABCD (8-23)

2×Quick TaqVF2VRMilliQTotal
25112350

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 1.5min
→30cycles

Gel extraction

pspA-DT 10.2μg/mL 1.42(260/280) 0.76(260/230)

Ligation

pspA-DT(Xba1, Pst1)LacP(pSB3C5)(Spe1, Pst1)Ligation High Ver.2total
5139

at 16℃

Electrophoresis

IMG 2140.jpg

LacP-torA-GFP-DT(8-15)

IMG 2141.jpg

LacP-torA-GFP-DT(9-23)

Ligation

LacP-torA-GFP-DT(EcoR1, Spe1)DT 17.2μg/mL(Xba1, Pst1)Ligation High Ver.2total
15252060

Liquid culture

  • J23107-tatABCD-8
  • J23107-tatABCD-20
  • LacP-torA-GFP-DT-21

September 6

Miniprep

  • J23107-tatABCD-8 82.4μg/mL 2.06(260/280) 2.95(260/230)
  • J23107-tatABCD-20 102.9μg/mL 2.04(260/280) 2.94(260/230)

Restriction

J23107-tatABCD 102.9μg/mLSpe1Pst1BufferHBSAMilliQtotal
100.50.530.515.530

at 37℃ for 1h

Transformation

LacP-pspA-DT(pSB3C5)

DNACompetent celltotal
22022

Gel extraction

IMG 2142.jpg

J23107-tatABCD(Spe1, Pst1) →We failed gel extraction because we mistook steps of experiment.

Electrophoresis

IMG 2143.jpg

LacP-torA-GFP-DT-pspA-DT

Restriction

by Terasaka

J23107-tatABCDSpe1Pst1BufferHBSAMilliQtotal
100.50.530.515.530

Gel extraction

IMG 2144.jpg

J23107-tatABCD(Spe1, Pst1) →See "September 7 -Gel extraction-"

September 7

Liquid culture

  • LacP-torA-GFP-DT-1 (CP resistance)
  • LacP-torA-GFP-DT-21 (CP resistance)

preculture for 1 hour

Colony PCR

LacP-torA-GFP-DT (1-8) LacP-pspA-DT (1-8)

2×Quick TaqVF2VRMilliQTotal
25112350

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 1.5min
→30cycles

Restriction

by Terasaka

J23107-tatABCDEcoR1Spe1BufferMBSAMilliQtotal
100.50.530.515.530
GFP-DTXba1Pst1BufferMBSAMilliQtotal
150.50.530.510.530

Electrophoresis

IMG 2145.jpg

① -⑧. LacP-pspA-DT
1-8. LacP-torA-GFP-DT

Gel extraction

IMG 2146.jpg
  • J23107-tatABCD(EcoR1, Spe1)
  • J23107-tatABCD(Pst1, Spe1) ①57.6μg/mL ②23.4μg/mL
  • GFP-DT(Xba1, Pst1)

Restriction

pspA-DT(145.5μg/mL)Xba1Pst1BufferMBSAMilliQtotal
200.50.530.55.530

37℃, overnight

Liquid culture

  • LacP-torA-GFP-DT-7 (LB 2mL)

+IPTG 0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM, 0.3mM

  • LacP-torA-GFP-DT-7 (LB 5mL)

September 8

Check the effect of Amp

We used 5mL of the liquid culture medium with LacP-torA-GFP-DT-7.
→See "September 7 -Liquid culture-"
We diluted it with LB until OD600=0.444, and dispensed it.
The dispense volume was 1.3mL.
We added 0/13/130μL of Amp(50mg/mL) to it.

5.5h after incubating at 37℃
Amp0μL13μL130μL
OD6002.4572.6960.310

Observation through a confocal microscope

We used the liquid culture media with LacP-torA-GFP-DT + 13μL of Amp(50mg/mL) + IPTG 0/0.1/0.15mM.
Their OD600 was 0.47.
3h after incubating at 37℃, we eliminated each supernatant using a centrifuge, and diluted them with LB to which Amp was added.
2.5h after incubating at 37℃, we observed them through a confocal microscope.
We failed to observe it.

Gel extraction

pspA-DT -5.5μg/mL

IMG 2147.jpg

Restriction

pspA-DT-3(322μg/mL)Xba1Pst1BufferMBSAMilliQtotal
1011331230

at 37℃ for 1h

September 9

Restriction

J23107-tatABCD(102.9μg/mL)EcoR1Spe1BufferMMilliQtotal
101131530

Electrophoresis

IMG 2148.jpg

1. J23107-tatABCD(EcoR1, Spe1)
2. J23107-tatABCD(before restriction)
3. pspA-DT(Xba1, Pst1)
4. pspA-DT(before restriction)

Gel extraction

IMG 2149.jpg

  • J23107-tatABCD(EcoR1, Spe1) 26μg/mL
  • pspA-DT(Xba1, Pst1) 199μg/mL

Liquid culture

  • J23100/J23101/J23106-1
  • pspA-DT-5 ×2
  • LacP-torA-GFP-DT-21
  • GFP generator-2
  • LacP-pspA-DT-6

September 10

Miniprep

  • J23100-1 105.5μg/mL
  • J23101-1 76.0μg/mL
  • J23106-1 90.9μg/mL
  • pspA-DT-5 ①104.1μg/mL ②80.0μg/mL
  • LacP-torA-GFP-DT-21 79.6μg/mL
  • GFP generator-2 84.1μg/mL
  • LacP-pspA-DT-6 99.8μg/mL

Ligation

  • J23107-tatABCD-DT
J23107-tatABCD(EcoR1, Spe1)DT(EcoR1, Xba1)Ligation High Ver.2total
1057.522.5
  • Negative control for J23107-tatABCD-DT
DT(EcoR1, Xba1)Ligation High Ver.2total
5510
  • J23107-tatABCD-pspA-DT
J23107-tatABCD(Spe1, Pst1)pspA-DT(Xba1, Pst1)Ligation High Ver.2total
36918
  • Negative control for J23107-tatABCD-pspA-DT
J23107-tatABCD(Spe1, Pst1)Ligation High Ver.2total
336

Restriction

IMG 2151.jpg
DNASpe1Pst1BufferHMilliQtotal
151131030

DNA=J23100(105.5μg/mL), J23101(76.0μg/mL), J23106(90.9μg/mL)
at 37℃

Gel extraction

IMG 2152.jpg
IMG 2153.jpg

J23100 27.1μg/mL
J23101 50.2μg/mL
J23106 16.1μg/mL

Ligation

  • J23100/J23101/J23106-kil-DT
J23100/J23101/J23106(Spe1, Pst1)kil-DT(Xba1, Pst1)Ligation High Ver.2total
1539
  • Negative control for J23100/J23101/J23106-kil-DT
J23100/J23101/J23106(Spe1, Pst1)MilliQLigation High Ver.2total
1539

Transformation

J23100/J23101/J23106-kil-DT

September 11

Colony PCR

  • J23107-tatABCD-DT (1-8)
  • J23107-tatABCD-pspA-DT (1-8)
2×Quick TaqVF2VRMilliQTotal
25112350

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 3.5min
→30cycles

Electrophoresis

IMG 2155.jpg
IMG 2156.jpg
  • J23107-tatABCD-DT (1-8)
  • J23107-tatABCD-pspA-DT (1-8)

Restriction

IMG 2154.jpg
GFP generator(84.1μg/mL)Xba1Pst1BufferMBSAMilliQtotal
151133730
LacP-torA-GFP-DT(85.0μg/mL)Spe1Pst1BufferHMilliQtotal
151131030
LacP-pspA-DT(99.8μg/mL)Xba1Pst1BufferMBSAMilliQtotal
151133730

Liquid culture

pSB1C3(self-ligation) ×5 in LB 3mLCP 3μL
When OD600=approximately 0.6, we added 0/30/60/150/300μL of Amp(50mg/mL) to each liquid culture medium.
1h and 5h after incubating at 37℃, we measured OD600.

1h5h
0-0.0200.403
30-0.0460.544
600.2630.007
1500.0610.839
3000.0931.441

Ligation

pSB3C5(EcoR1, Pst1)pspA-DT(Xba1, Pst1)J23107-tatABCD(EcoR1, Spe1)Ligation High Ver.2total
1354.513.5

4℃, overnight

Colony PCR

  • J23100-kil-DT (1-6)
  • J23101-kil-DT (1-7)
  • J23106-kil-DT (1-2)
2×Quick TaqVFVRMilliQTotal
23112550

Gel extraction

IMG 2159.jpg
  • GFP generator (Xba1, Pst1) 14.1μg/mL
  • LacP-torA-GFP-DT (Spe1, Pst1) 42.9μg/mL 1.13(260/280) 1.19(260/230)
  • LacP-pspA-DT (Xba1, Pst1) 20.0μg/mL 1.01(260/280) 1.30(260/230)

Electrophoresis

IMG 2160.jpg
  • J23107-tatABCD-DT (1-8)
  • J23106-kil-DT (1, 2)
  • J23100-kil-DT (1-4)

Liquid culture

  • LacP-torA-GFP-DT
  • pSB1C3

Colony PCR

We did PCR 10 more cycle to amplify products of PCR on 9/11 of J23107-tatABCD-pspA-DT and J23107-tatABCD-DT.



September 12

Colony PCR

J23107-tatABCD-pspA-DT (9-15) 94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 4min
→26cycles

Check the effect of Amp

We cultured pSB1C3 for overnight.
→See "September 7 -Liquid culture-"
We diluted it with LB, and dispensed it.
We added Amp(50mg/mL) to it until density of Amp was 1/10, 1/30, 1/50 .

Ampstart5 hours
1/100.5570.077
1/300.6280.166
1/500.4720.055
00.5942.585

Electrophoresis

IMG 2162.jpg
  • J23107-tatABCD-pspA-DT
IMG 2161.jpg
  • J23101-kil-DT (1-7)

Restriction

pspA(pSB3C5) (106μg/mL)EcoR1Pst1BufferHMilliQtotal
151131030
pspA-DT-3Xba1Pst1BufferMBSAMilliQtotal
151133730
J23107-tatABCD (102.4μg/mL)EcoR1Spe1BufferMMilliQtotal
151131030

Electrophoresis

IMG 2165.jpg
  • pspA(pSB3C5)(EcoR1, Pst1)
  • pspA-DT(Xba1, Pst1)
  • J23107-tatABCD(EcoR1, Spe1)
  • pSB1C3(EcoR1, Spe1)

Transformation

J23107-tatABCD-pspA-DT(pSB3C5)

DNACompetent celltotal
22022

on ice for 30 minites heatshock at 42℃ for 1 hour on ice 2 minites then add SOC medium 200μl preculture at 37℃ for 1 hour incubate CP culture plate

Gel extraction

IMG 2166.jpg
  • pspA(pSB3C5)(EcoR1, Pst1) 26.8μg/mL 1.27(260/280) 0.32(260/230)
  • pspA-DT(Xba1, Pst1) 26.1μg/mL 1.13(260/280) 0.55(260/230)
  • J23107-tatABCD(EcoR1, Spe1) 1.5μg/mL 1.99(260/280) 0.06(260/230)

Ligation

pSB3C5(EcoR1, Pst1) 26.8μg/mLpspA-DT(Xba1, Pst1) 26.1μg/mLJ23107-tatABCD(EcoR1, Spe1) 1.5μg/mLLigation High Ver.2total
136515
pSB3C5(EcoR1, Pst1) 26.8μg/mLMilliQLigation High Ver.2total
19515
LacP(pSB3C5)(Spe1, Pst1) 10μg/mLGFP-DT(Xba1, Pst1) 7.0μg/mLLigation High Ver.2total
1539
LacP(pSB3C5)(Spe1, Pst1) 10μg/mLMilliQLigation High Ver.2total
1539

4℃, overnight

Observation through a confocal microscope

Evernoteに上がっている写真をお願いします。

September 13

Miniprep

  • J23107-tatABCD-pspA-DT(pSB3C5)-1 130μg/mL 1.37(260/280) 0.79(260/230)


Restriction

J23107-tatABCD-pspA-DT(pSB3C5) (130μg/mL)Spe1Pst1BufferHMilliQtotal


101131530

Transformation

  • LacP-GFP generater
  • LacP
  • J23107-tatABCD-pspA-DT(pSB3C5)
  • J23107-tatABCD

Colony PCR

J23107-tatABCD-pspA-DT

→See "September 14 -Colony PCR-"

September 14

Colony PCR

IMG 2169.jpg

See "September 13 -Colony PCR-"
J23107-tatABCD-pspA-DT

Colony PCR

  • J23107-tatABCD-pspA-DT (1-6)
2×Quick TaqVFVRMilliQTotal
23112550

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 4min
→30cycles

September 15

Liquid culture

  • J23107-tatABCD-pspA-DT-1
  • J23107-tatABCD-pspA-DT-2
  • J23107-tatABCD-pspA-DT-3

Miniprep

  • J23107-tatABCD-pspA-DT-1 22.9μg/mL
  • J23107-tatABCD-pspA-DT-2 51.8μg/mL 1.57(260/280) 1.01(260/230)
  • J23107-tatABCD-pspA-DT-3 28.5μg/mL 1.57(260/280) 0.94(260/230)

Restriction

J23107-tatABCD-pspA-DT (51.8μg/mL)EcoR1Spe1BufferMMilliQtotal
150.50.531130
LacP-torA-GFP-DT (79.6μg/mL)EcoR1Xba1BufferMBSAMilliQtotal
100.50.530.515.530
LacP-kil-DT (56.0μg/mL)EcoR1Xba1BufferMBSAMilliQtotal
150.50.530.510.530
J23107-tatABCD-pspA-DT (51.8μg/mL)EcoR1Pst1BufferMMilliQtotal
150.50.531130

37℃, 2 hours

Electrophoresis

IMG 2170.jpg

①. 1kb ladder
②. J23107-tatABCD-pspA-DT
③. J23107-tatABCD-pspA-DT(EcoR1, Spe1)
④. J23107-tatABCD-pspA-DT(EcoR1, Pst1)
⑤. LacP-kil-DT
⑥. LacP-kil-DT(EcoR1, Xba1)
⑦. LacP-torA-GFP-DT
⑧. LacP-torA-GFP-DT(EcoR1, Xba1)

Gel extraction

IMG 2171.jpg
IMG 2172.jpg

①. 1kb ladder
②. J23107-tatABCD-pspA-DT(EcoR1, Spe1)
③. J23107-tatABCD-pspA-DT(EcoR1, Spe1)
④. J23107-tatABCD-pspA-DT(EcoR1, Pst1)
⑤. J23107-tatABCD-pspA-DT(EcoR1, Pst1)
⑥. 1kb ladder

  • J23107-tatABCD-pspA-DT(EcoR1, Spe1) 14.2μg/mL 1.28(260/280) 0.54(260/230)
  • J23107-tatABCD-pspA-DT(EcoR1, Pst1) 7.3μg/mL 1.55(260/280) 0.38(260/230)

Purification

  • LacP-torA-GFP-DT(EcoR1, Xba1) 7.7μg/mL 1.41(260/280) 0.99(260/230)
  • LacP-kil-DT(EcoR1, Xba1) →failed

Ligation

LacP-torA-GFP-DT(EcoR1, Xba1)J23107-tatABCD-pspA-DT(EcoR1, Spe1)Ligation High Ver.2total
410721
pSB1C3(EcoR1, Pst1)J23107-tatABCD-pspA-DT(EcoR1, Pst1)Ligation High Ver.2total
410721

at 16℃ for overnight

September 16

Transformation

LacP-torA-GFP-DT-J23107-tatABCD-pspA-DT
J23107-tatABCD-pspA-DT(pSB1C3)

September 17

Colony PCR

J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (1-6)
J23107-tatABCD-pspA-DT(pSB1C3) (1-6)
J23107-kil-DT (6-10)

Restriction

LacP-torA-GFP-DT (126μg/mL)EcoR1Xba1BufferMBSAMilliQtotal
1011331230
LacP-torA-GFP-DT (126μg/mL)Xba1BufferMBSAMilliQtotal
31332030

Electrophoresis

IMG 2173.jpg

A1-A6. J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (1-6)
B1-B6. J23107-tatABCD-pspA-DT(pSB1C3) (1-6)

Liquid culture

  • J23107-tatABCD-pspA-DT(pSB1C3)
  • J23107-tatABCD-pspA-DT(pSB3C5)
  • LacP-torA-GFP-DT(pSB3C5)

Electrophoresis

写真お願いします。
lane1. 1kb ladder
lane2. J23101-kil-DT-6
lane3. J23101-kil-DT-7
lane4. J23101-kil-DT-8
lane5. J23101-kil-DT-10
lane6. LacP-torA-GFP-DT
lane7. LacP-torA-GFP-DT(EcoR1, Xba1)
lane8. LacP-torA-GFP-DT(Xba1)

Miniprep

  • J23106-kil-DT 11.7μg/mL 1.93(260/280) 1.46(260/230)

September 18

Gel extraction

IMG 2174.jpg
  • LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mL 1.37(260/280) 0.82(260/230)

Ligation

J23107-tatABCD-pspA-DT(EcoR1, Spe1) 14.2μg/mLLacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mLLigation High Ver.2total
81.59.519
LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mLLigation High Ver.2total
1.51.53

at 4℃ for 1 hour

Transformation

  • J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT
  • LacP-torA-GFP-DT(EcoR1, Xba1)

Observation through a confocal microscope

We made sample for observation through a confocal microscope.
We incubated sample with 0mM/0.5mM IPTG for 1.5 hours.
Then, we incubated onto medium without IPTG for 2.5 hours.

IPTG(mM)medium
0LB
0.1LB
0.1LB with 0.1mM IPTG (positive control)
0LB with 2 percent glucose
0.1LB with 2 percent glucose
0LB with 5 percent glucose
0.1LB with 5 percent glucose
0LB with 2 percent glucose (make medium new after incubate for 1.5 hours)
0LB with 2 percent glucose (make medium new after incubate for 1.5 hours)

Restriction

pSB4K5EcoR1Pst1BufferHMilliQtotal
101131530
pSB4K5EcoR1BufferHMilliQtotal
30.515.530
pSB4K5Pst1BufferHMilliQtotal
30.515.510
LacP-torA-GFP-DTXba1Pst1BufferMBSAMilliQtotal
1011331230
LacP-torA-GFP-DTXba1BufferMBSAMilliQtotal
2111510
LacP-torA-GFP-DTPst1BufferHMilliQtotal
211610

37℃, 2 hours

Gel extraction

  • LacP-torA-GFP-DT(Xba1, Pst1) 9.7μg/mL 1.34(260/280) 0.62(260/230)
  • pSB4K5(EcoR1, Pst1) 9.6μg/mL 1.28(260/280) 0.32(260/230)

Miniprep

  • J23107-tatABCD-psp-DT(pSB1C3) 78μg/mL
  • J23107-tatABCD-psp-DT(pSB1C3) 48μg/mL
  • LacP-torA-GFP-DT 29μg/mL
  • J23101-kil-DT 25μg/mL

Restriction enzyme processing

J23101-Kil-DT 25µg/mlBufferMXba1Pst1BSAMilliQtotal
406116660
J23101-Kil-DT 25µg/mlBufferMXba1BSAMilliQtotal
310.514.510
J23101-Kil-DT 25µg/mlBufferHPst1MilliQtotal
310.55.560

at 37℃ for 2 hours

Electrophoresis

写真お願いします。
①. pSB4K5
②. pSB4K5(Pst1)
③. pSB4K5(EcoR1)
④. pSB4K5(EcoR1, Pst1)
⑤. LacP-torA-GFP-DT
⑥. LacP-torA-GFP-DT(Xba1)
⑦. LacP-torA-GFP-DT(Pst1)
⑧. LacP-torA-GFP-DT(Xba1, Pst1)

写真お願いします。
①. 1kb ladder
②. J23101-Kil-DT(Xba1, Pst1)
③. J23101-Kil-DT(Xba1, Pst1)
④. J23101-Kil-DT(Xba1, Pst1)
⑤. J23101-Kil-DT(Xba1, Pst1)
⑥. 1kb ladder


Restriction enzyme processing

J23101-Kil-DTBufferMXba1Pst1BSAMilliQtotal
153113730
J23101-Kil-DTBufferMXba1BSAMilliQtotal
310.514.510
J23101-Kil-DTBufferHPst1MilliQtotal
310.55.510

37℃

September 19

GeneClean II

LacP-torA-GFP-DT
4k5

Ligation

LacP-torA-GFP-DT(Xba1,Pst1) 9.7μg/mLpSB4K5(EcoR1,Pst1) 9.6μg/mLJ23107-tatABCD-PsPA-DT(EcoR1,Spe1) 14.2μg/mLLigation High Ver.2total
727824μL
pSB4K5(EcoR1,Pst1) 9.6μg/mLLigation High Ver.2MilliQtotal
212124μL

incubate at 37℃ for 2h

Electrophoresis

J23101-kil-DT(Xba1,Pst1) (Xba1) (Pst1)
Lane1: 1kb ladder
Lane2: empty
Lane3: plasmid
Lane4: Xba1
Lane5: Pst1
Lane6: Xba1,Pst1

Liquid Culture

We did liquid culture J23106-kil-DT(9/10, Amp)'s 6,7 at LB mediumAmp.

Transformation

J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT
pSB4K5 negative control

Liquid culture

J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT:1~5

Colony PCR

J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT

VF2VRQuick tagMilliQtotal
11252350

Predenature 94℃ 2min
Denature 94℃ 30sec
Annealing 55℃ 30sec
Extension 68℃ 5min
→30cycles

Restriction enzyme processing

LacP-kil-DT(4) 62.5μg/mLXba1Pst110xBSAMilliQBufferMtotal
1011312330
LacP-kil-DT(4)EcoR1Pst1MilliQBufferHtotal
101115330
LacP-kil-DTXba110xBSAMilliQBufferMtotal
20.515.5110
LacP-kil-DTEcoR1MilliQBufferHtotal
20.56.5110
LacP-kil-DTPst1MilliQBufferHtotal
20.56.5110

at 37℃, 2 hours

Electrophoresis

Lane1: 1kb ladder
Lane2: empty
Lane3: plasmid
Lane4: EcoR1
Lane5: Xba1
Lane6: Pst1
Lane7: EcoR1,Pst1
Lane8: Xba1,Pst1

Electrophoresis

J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT
Lane1: ladder
Lane2: 1
Lane3: 2
Lane4: 3
Lane5: 4
Lane6: 5

Ligation

LacP-torA-GFP-DT(Xba1,Pst1) 9.7μg/mLpSB4K5(EcoR1,Pst1) 9.6μg/mLJ23107-tatABCD-PsPA-DT(EcoR1,Spe1) 14.2μg/mLLigation High Ver.2total
747927μL

(Negative control)

pSB4K5(EcoR1,Pst1) 9.6μg/mLLigation High Ver.2MilliQtotal
491427μL

Restriction enzyme processing

torA-R9 101.8μg/mLbufferHMilliQEcoR1Pst1total
103151130
torA-R9 101.8μg/mLbufferHMilliQEcoR1Pst1total
315.50.5010
torA-R9 101.8μg/mLbufferHMilliQEcoR1Pst1total
315.500.510

PCR

torA-R9MilliQ10xPCR buffer for KOD plus neo2mM dNTP325mM MgSO4M13KOD plus neototal
0.5345531.5150

Predenature 94℃ 2min
Denature 98℃ 10sec
Annealing 60℃ 30sec
Extension 68℃ 20sec
→35cycles

Electrophoresis

torA-R9,plasmid,(EcoR1),(Pst1),(EcoR1,Pst1)
Lane1: 1kb ladder
Lane2: empty
Lane3: plasmid
Lane4: EcoR1
Lane5: Pst1
Lane6: EcoR1,Pst1
Lane7: empty
Lane8: 1kb ladder

Liquid culture

LacP-tatA-GFP-DT (9/4)

PCR purification

torA-R9 68.0μg/mL

September 20

Gel extraction

LacP-kil-DT (EcoR1,Pst1) (Xba1,Pst1)

Restriction enzyme processing

torA-R9 68.0μg/mLbufferMSpe1MilliQTotal
2031630

at 37℃, 2h

Ligation

LacP-kil-DT(Xba1,Pst1) 12.4μg/mLpSB1C3(EcoR1,Pst1) 34.2μg/mLJ23107-tatABCD-PsPA-DT(EcoR1,Spe1) 14.2μg/mLLigation High Ver.2total
747927μL
16℃, 1h

(Negative control)

pSB1C3(EcoR1,Pst1) Ligation High Ver.2MilliQtotal
491427μL

Transformation

Using above Ligation and Negative control's product and torA-R9.

Miniprep

J23106-kil-DT 6; 12.0μg/mL, (260/280): 1.78, (260/230): 1.73
J23106-kil-DT 7; 13.6μg/mL, (260/280): 1.85, (260/230): 1.73

Restriction enzyme processing

GFP-DT 84.1μg/mLbufferM10xBSABbaIMilliQtotal
15331830

at 37℃, 2 hours

Colony PCR

We made J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT's 1~8 samples.

Quick tagtat f primerVRMilliQtotal
25112350

Predenature 94℃ 2min
Denature 94℃ 30sec
Annealing 54℃ 30sec
Extension 68℃ 4min
→30cycles

Liquid culture

We did liquid culture J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT's 1~4 samples.

Electrophoresis

J23701-tatABCD-pspA-DT-LacP-torA-GFP-DT Lane1: 1kb ladder
Lane2: 1
Lane3: 2
Lane4: 3
Lane5: 4
Lane6: 5
Lane7: 6
Lane8: 7
Lane9: 8
Lane10: 1kb ladder
Lane11: empty
Lane12: empty
torA-R9 (EcoR1,Pst1): 31μg/mL, (280/280): 0.80, (280/230): 0.39
pSB1C3(EcoR1,Pst1): 22μg/mL, (280/280):1.33, (280/230):0.6
torA-R9 (Spe1): 56μg/mL, (280/280): 1.11, (280/230): 0.69
GFP-DT(Xba1): 45μg/mL, (280/280): 1.15, (280/230): 0.88

Ligation

torA-R9 (EcoR1,Pst1)pSB1C3(EcoR1,Pst1)Ligation High Ver.2total
82515μL
torA-R9 (Spe1)pSB1C3(Xba1)Ligation High Ver.2total
3339μL

at 16℃, 1hour

Colony PCR

J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (4k5)
numbering 9~15

Transformation

J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (4k5)
J23107-tatABCD-pspA-DT-LacP-torA-kil-DT(pSB1C3)
Negative control (pSB1C3)
Negative control(pSB4K5)
torA-R9(pSB1C3)

Liquid culture

J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT: colony PCR(9~15)

2xQuick tagVFVRMilliQtotal
25112350

Predenature 94℃ 2min
Denature 94℃ 30sec
Annealing 55℃ 30sec
Extension 68℃ 5min
→30cycles

PCR

torA-R9-GFP-DTMilliQKOD plus ver.2 bufferdNTP3MgSO4M13VRKOD plustotal
1355521.51.5152

Predenature 94℃ 2min
Denature 94℃ 15sec
Annealing 55℃ 30sec
Extension 68℃ 5min
→30cycles

Miniprep

J23701-tatABCD-pspA-DT-LacP-torA-GFP-DT:2,3
2: 10.4μg/mL, (250/280): 1.45, (260/230): 0.95
3: 6.0μg/mL, (250/280): 1.32, (260/230): 0.79
(*)100μL

Electrophoresis

torA-R9-GFP-DT(after PCR)

September 21

Gel extraction

torA-R9-GFP-DT(after PCR): 10.3μg/mL, (260/280): 1.28, (220/230): 0.49

Electrophoresis

J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(kan): 9~15

Electrophoresis

J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT: 1,2
Lane1: empty
Lane2: empty
Lane3: 2
Lane4: 3
Lane5: 1kb ladder

Restriction enzyme processing

LacP-kil-DT(3)Xba1Pst110xBSAMilliQBufferMtotal
151137330
LacP-torA-GFP-DP(9/5)Xba1Pst110xBSAMilliQBufferMtotal
151137330
LacP-kil-DTXba110xBSAMilliQBufferMtotal
20.515.5110
LacP-torA-GFP-DPXba110xBSAMilliQBufferMtotal
20.515.5110
LacP-kil-DTPst1MilliQBufferHtotal
20.56.5110
LacP-torA-GFP-DPXba1Pst1MilliQBufferHtotal
20.516.5110
J23107-tatABCD-pspA-DTSpe1Pst1BufferHMilliQtotal
201141440
J23107-tatABCD-pspA-DTSpe1BufferMMilliQtotal
20.516.510
J23107-tatABCD-pspA-DTPst1BufferHMilliQtotal
20.516.510

at 37℃, 2hours

September 22

Miniprep

torA-R9(Amp)1: 113.7μg/mL, (260/280): 1.96, (280/230): 2.42
torA-R9(Amp)2: 97.8μg/mL, (260/280): 1.67, (280/230): 1.72
torA-R9(Amp)3: 104.2μg/mL, (260/280): 1.93, (280/230): 2.67
LacP-torA-GFP-DT 6: 37.4μg/mL, (260/280): 2.24, (280/230): 3.27

Electrophoresis

LaneA: J23107-tatABCD (9/6)
LaneB: J23107-tatABCD-pspA-DT pSB3C5 (9/13)
LaneC: J23107-tatABCD-pspA-DT 1C5 (9/15)
LaneD: J23107-tatABCD-pspA-DT (9/15)
LaneE: J23107-tatABCD-pspA-DT(pSB1C3) (9/18)
LaneF: J23107-tatABCD-pspA-DT(pSB3C5) (9/18)
LaneG: J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT:2 (9/20)
LaneH: J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT:3 (9/20)
LaneA~H are (EcoR1,Pst1)

Restriction enzyme processing

J23107-tatABCD-pspA-DT-LacPtorA-GFP-DT:2BufferHPst1MilliQtotal
15311230
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT:3BufferHPst1MilliQtotal
15311230

Restriction enzyme processing

LacP-torA-GFP-DT(37.4μg/mL)BufferHEcoR1Pst1total
2531130

Electrophoresis

1: J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT:2
2: LacP-torA-GFP-DT (EcoR1,Pst1) (9/22)
3: J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT:3

September 23

Electrophoresis

写真お願いします。

  • LacP-torA-GFP-DT(EcoR1, Pst1)

lane_1 1kb ladder lane_3 DNA lane_4 DNA lane_5 DNA

Gel extraction

写真お願いします。

  • J23107-tatABCD-pspA-DT(pSB1C3)(Spe1, Pst1)
  • LacP-torA-GFP-DT(Xba1, Pst1)

lane_1 1kb ladder
lane_3 J23107-tatABCD-pspA-DT(pSB1C3)(Spe1, Pst1)
lane_5 LacP-torA-GFP-DT(Xba1, Pst1)

Restriction

LacP-torA-GFP-DT (78.4μg/mL)Xba1Pst1BufferMBSAMilliQtotal
151133730
LacP-torA-GFP-DT (78.4μg/mL)Xba1BufferMBSAMilliQtotal
30.5114.510
J23107-tatABCD-pspA-DT(pSB1C3) (78μg/mL)Spe1Pst1BufferHMilliQtotal
9112720

Ligation

LacP-torA-GFP-DT(EcoR1, Pst1) (5.5μg/mL)pSB4K5 (9.6μg/mL)Ligation High Ver.2total
84618

at 16℃ for 1 hour

Liquid culture

  • LacP-tatABCD-pspA-DT (2, 3, 5, 6)

Colony PCR

LacP-tatABCD-pspA-DT (1-6)

2×Quick TaqVF2VRMilliQTotal
25112350

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 4min
→30cycles

Electrophoresis

写真お願いします。
①, LacP-torA-GFP-DT
②, LacP-torA-GFP-DT(Xba1, Pst1)
③, LacP-torA-GFP-DT(Xba1)
④, LacP-torA-GFP-DT(Pst1)
⑤, J23107-tatABCD-pspA-DT(Spe1, Pst1)

Liquid culture

  • LacP-torA-GFP-DT (4, 7)
  • LacP-GFP generater (1-3)
  • T7-6His-GFP-DT (1, 2)

Gel extraction

写真お願いします。

  • LacP-torA-GFP-DT (Xba1, Pst1) 6.8μg/mL 1.27(260/280) 0.40(260/230)

Purification

  • J23107-tatABCD-pspA-DT(Spe1, Pst1) 15.1μg/mL 1.49(260/280) 0.80(260/230)

Ligation

J23107-tatABCD-pspA-DT(Spe1, Pst1) (15.1μg/mL)LacP-torA-GFP-DT (Xba1, Pst1) (6.8μg/mL)Ligation High Ver.2total
104721
J23107-tatABCD-pspA-DT(Spe1, Pst1) (15.1μg/mL)LacP-kil-DT (Xba1, Pst1) (12.4μg/mL)Ligation High Ver.2total
82515

at 16℃ for 1 hour

Transformation

LacP-torA-GFP-DT(pSB4K5)
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT
J23107-tatABCD-pspA-DT-LacP-kil-DT

DNAcompetent celltotal
21012

preculture at 37℃ for 1 hour culture at 37℃ for 16 hours

Electrophoresis

写真お願いします。
J23107-tatABCD-pspA-DT(pSB1C3) (1-6)
lane_1 1kb ladder lane_2~7 DNA lane_8 1kb ladder

September 24

Miniprep

  • J23107-tatABCD-pspA-DT-2 139.4μg/ml 1.69(260/280) 1.83(260/230)
  • J23107-tatABCD-pspA-DT-3 134.7μg/ml 1.61(260/280) 1.69(260/230)
  • J23107-tatABCD-pspA-DT-5 139.3μg/ml 1.55(260/280) 1.45(260/230)
  • J23107-tatABCD-pspA-DT-6 138.5μg/ml 1.58(260/280) 1.40(260/230)
  • LacP-torA-GFP-DT-4
  • LacP-torA-GFP-DT-7
  • T7-6His-GFP-DT-1
  • T7-6His-GFP-DT-2

Colony PCR

LacP-GFP generater (1-3)

2×Quick TaqVF2VRMilliQTotal
25112350

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 1min
→30cycles

写真お願いします。
①-③, LacP-GFP generater (1-3)

Liquid culture

  • J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (1-4)
  • J23107-tatABCD-pspA-DT-LacP-kil-DT (1-3)

Colony PCR

J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (1-4)
J23107-tatABCD-pspA-DT-LacP-kil-DT (1-3)

2×Quick TaqVF2VRMilliQTotal
25112350

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 5min
→30cycles

Electrophoresis

写真お願いします。
lane1, 1kb ladder
lane2-5, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT (1-4)
lane6-8, J23107-tatABCD-pspA-DT-LacP-kil-DT (1-3)

September 25

Liquid culture

  • J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB4K5)-2

Miniprep

J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3)-3: 26.2μg/mL, (260/280): 1.50, (280/230): 0.65
J23107-tatABCD-pspA-DT-LacP-torA-GFP(pSB1C3)-3: 24.8μg/mL, (260/280): 1.56, (280/230): 0.76
LacP-GFP generater-1: 42.0μg/mL, (260/280): 1.58, (280/230): 0.94

Restriction enzyme processing

J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (26.2ng/µL)EcoRlSpelbuffer MMiliQTotal
151131030
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (26.2ng/µL)EcoRlbuffer MMiliQTotal
20.516.510
J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (26.2ng/µL)Spelbuffer MMiliQTotal
20.516.510
J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (24.8ng/µL)EcoRlPstlbuffer HMiliQTotal
151131030
J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (24.8ng/µL)Pstlbuffer HMiliQTotal
20.516.510
J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (24.8ng/µL)EcoRlbuffer HMiliQTotal
20.516.510

at 37℃ for 2 hours

Electrophoresis

写真お願いします。
lane1, 1kb ladder
lane2, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3)
lane3, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl, Spel)
lane4, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl)
lane5, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (Spel)
lane6, J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) lane7, J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (EcoRl, Pstl)
lane8, J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (EcoRl)
lane9, J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (Pstl)
lane10, LacP-torA-GFP-DT(EcoR1,Xba1)(9/15)
lane11, LacP-torA-GFP-DT(EcoR1,Xba1)(9/17)
lane12, 1kb ladder

→The bands didn't appear, so we did one more electrophoresis.
写真お願いします。
lane1, 1kb ladder
lane2, LacP-torA-GFP-DT(EcoR1,Xba1)(9/17)
lane3, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3)
lane4, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl, Spel)
lane5, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl)
lane6, J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (Spel)

Liquid culture

  • LacP-torA-GFP-DT(pSB4K5) (1-4)

Restriction enzyme processing

J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3)EcoRlSpelbuffer MMiliQTotal
151131030
J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3)EcoRlPstlbuffer HMiliQTotal
151131030

at 37℃ for 1 hour

Electrophoresis

J23107-tatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl, Spel)
J23107-tatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (EcoRl, Pstl)
→The bands didn't appear.



Retrieved from "http://2012.igem.org/Team:Kyoto/Secretion/Notebook"