Team:Kyoto/Secretion/Notebook
From 2012.igem.org
(→February 22) |
(→Colony PCR) |
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Line 1,415: | Line 1,415: | ||
68℃, 1min<br> | 68℃, 1min<br> | ||
→35cycles<br><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 | ||
+ | |} | ||
+ | {|class="wikitable" | ||
+ | !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==== | ||
+ | 写真貼ってください | ||
+ | =====① 1kb ladder===== | ||
+ | =====②, ③ MgSO4 3μL, pspA===== | ||
+ | =====④, ⑤ MgSO4 5μL, pspA===== | ||
+ | =====⑥ negative control(MilliQ)===== | ||
+ | =====⑧ 1kb ladder===== | ||
+ | ==August 18== | ||
+ | ==August 19== | ||
+ | ==August 20== | ||
+ | |||
+ | |||
+ | |||
+ | |||
{{Kyoto/footer}} | {{Kyoto/footer}} |
Revision as of 05:15, 21 September 2012
Secretion Notebook
February 7
Preculture
by_???
We started preculture at 12:10.
February 20
February 21
February 22
PCR
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
98℃, 10sec
59℃, 30sec
68℃, 3min
→25cycles
Electrophoresis
Checking Dpn1
Buffer | lacp(28.7ng/μL) | Dpn1 | MilliQ | total |
---|---|---|---|---|
2 | 10 | 0.5 | 7.5 | 20 |
2 | 10 | 0 | 5 | 17 |
Gel extraction
lane 1 of the gel 45.0μg/mL
PCR purification
product 38.2μg/mL
PCR
TMAO reductase
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
98℃, 10sec
59℃, 30sec
68℃, 3min
→25cycles
Electrophoresis
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
February 23
Colony PCR
tatABCD(2 samples)
Buffer | dNTPs | MgSO4 | primer-f | primer-r | KOD plus | MilliQ | total |
---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |
Predenature 94℃ 1min
Denature 98℃ 10sec
Annealing 59℃ 30sec
Extension 68℃ 3min
→25cycles
Electrophoresis
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
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
Buffer2 | GFP2 | BSA | MilliQ | Dpn1 |
---|---|---|---|---|
3 | 3 | 0.3 | 23 | 1 |
Colony PCR
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
Denature 98℃,10sec
Annealing 59℃,30sec
Extension 68℃,3min
→25cycles
Electrophoresis
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.
pSB3C5-5 | pSB3C5-6 | pSB3C5-5(1% glucose) | pSB3C5-6(1% glucose) |
---|---|---|---|
8.5[µg/ml] | -1.8 | -17.9 | -18.2 |
PCR
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
Predenature 94℃,2min
Denature 98℃,10sec
Annealing 59℃,30sec
Extension 68℃,3min
→25cycles
February 28
Electrophoresis
1. 1kb ladder
2. PCR1 →Product of gel extraction : tatABCD with prefix and suffix 105[ng/µL]
3. PCR2
Restriction
Buffer2 | plasmid(?) | enzyme | MilliQ | total |
---|---|---|---|---|
2 | 2 | 0.2 | 15.8 | 20 |
incubate 1 hour at 37℃
Electrophoresis
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 Mix | primer-f | primer-r | template | MilliQ | total |
---|---|---|---|---|---|
25 | 1.0 | 1.0 | 0.5 | 22.5 | 50 |
Predenature 94℃,2min
Denature 94℃,30sec
Annealing 59℃,30sec
Extension 68℃,3min
→25cycles
Restriction
BufferH | tatABCD | EcoR1 | Spe1 | MilliQ | total |
---|---|---|---|---|---|
2 | 5 | 0.2 | 0.2 | 12.6 | 20 |
PCR purification
We eluted the product for 30µL MilliQ
Ligation
Insert(tatABCD) | Vector(pSB1C3) | Ligation High | total |
---|---|---|---|
10 | 1 | 5 | 16 |
4℃, overnight
February 29
Transformation
tatABCD | competent cell | total |
---|---|---|
1 | 10 | 11 |
Checking Restriction enzyme
plasmid seems to be 1-18C promoter | Enzyme | Buffer | MilliQ | total |
---|---|---|---|---|
2 | 0.2 | 2 | 15.8 | 20 |
Checking tatABCD
tatABCD | Hind3 | Buffer | MilliQ | total |
---|---|---|---|---|
5 | 0.2 | 2 | 12.8 | 20 |
PCR
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
Denature 98℃,10sec
Annealing 60℃,30sec
Extension 68℃,3min
→30cycles
Electrophoresis
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
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
98℃, 10sec
60℃, 30sec
68℃, 1.5min
→25cycles
- Kil
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
98℃, 10sec
61℃, 30sec
68℃, 1min
→20cycles
Electrophoresis
1. 1kb ladder
2. TMAO1 (gDNA)
3. TMAO2 (product of gel extraction)
4. Kil
PCR
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
98℃, 10sec
61℃, 30sec
68℃, 30sec
20cycles
→Purification 230ng/µL
Restriction
Kil | EcoR1 | Spe1 | BufferH | MilliQ | total |
---|---|---|---|---|---|
5 | 0.2 | 0.2 | 2 | 12.6 | 20 |
incubate at 37℃, for 1.5 hours
PCR Purification
Ligation
Kil | pSB1C3 | Ligation High | total |
---|---|---|---|
5 | 1 | 3 | 9 |
at 4℃, for overnight
March 2
Ligation
Kil | pSB1C3 | Ligation High Ver.2 | total |
---|---|---|---|
5 | 1 | 3 | 9 |
tatABCD | pSB1C3 | Ligation | total |
---|---|---|---|
10 | 1 | 5 | 16 |
at 16℃ for 1 hour
Transformation
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
Quick Taq | primer-r | primer-f | template | MilliQ | total |
---|---|---|---|---|---|
25 | 1 | 1 | 0.5 | 22.5 | 50 |
Electrophoresis
(27)
Restriction
pSB3C5-5 | EcoR1 | Pst1 | BufferH | BSA | MilliQ | total |
---|---|---|---|---|---|---|
20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |
at 37℃, for 2 hour
Electrophoresis
1. 1kb ladder 2µL
2. pSB3C5 5µL + 6×Loading Buffer 1µL
・product of gel extraction(about 2700bp)
-30.9µg/mL
Restriction
GFP1,2,3
GFP | EcoR1 | Pst1 | Buffer | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |
at 37℃, for 2.5 hours
Restriction
DT | EcoR1 | Xba1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.2 | 0.2 | 3 | 0.3 | 16.3 | 30 |
Constitutive Promoter | Spe1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
15 | 0.2 | 0.2 | 3 | 0.3 | 11.3 | 30 |
at 37℃, 2 hours
- J23117-1:135ng/µL, J23107-1:115ng/µL
- DT3→PCR Purification
- Promoter→Gel Extraction
Checking TMAO
something seems to be TMAO | Buffer2 | EcoR1 | MilliQ | total |
---|---|---|---|---|
10 | 2 | 0.5 | 7.5 | 20 |
at 37℃, for 1 hour
Electrophoresis
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 Taq | primer-f | primer-r | MilliQ | total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
Electrophoresis
(28)
March 3
PCR
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
98℃, 10sec
50℃, 30sec
68℃, 1min
→30cycles
Miniprep
March 4
Sequence of tatABCD
Quick Taq | primer-f | promer-r sequence | template | MilliQ | total |
---|---|---|---|---|---|
25 | 1 | 1 | 1 | 23 | 50 |
Quick Taq | primer-f sequence | primer-r | template | MilliQ | total |
---|---|---|---|---|---|
25 | 1 | 1 | 1 | 23 | 50 |
Colony PCR of TMAO
buffer | dNTPs | NgSO4 | primer-f | primerr-r | KOD plus | MilliQ | total |
---|---|---|---|---|---|---|---|
5 | 5 | 4 | 1.5 | 1.5 | 1 | 32 | 50 |
→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
TMAO | EcoR1 | BufferH | BSA | MilliQ | total |
---|---|---|---|---|---|
10 | 0.2 | 3 | 0.3 | 16.5 | 30 |
TMAO | Xba1 | Pst1 | BudderM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.2 | 0.2 | 3 | 0.3 | 16.3 | 30 |
at 37℃ for 1 hour
Electrophoresis
Transformation
pSB1C3 | competent cell(made at 2/8) | total |
---|---|---|
5 | 100 | 105 |
March 5
Restriction
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
→Then we did ethanol precipitation
Ligation
Kil(EcoR1, Spe1) | pSB1C3(EcoR1) | Ligation High | total |
---|---|---|---|
5 | 1 | 3 | 9 |
at 16℃ for 1 hour
Transformation
Kil | competent cell | total |
---|---|---|
1 | 10 | 11 |
We used commercially available competent cells in this time.
PCR
TMAO
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)
Restriction
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
1→Ethanol precipitation 45.4µg/mL
2→Gel extraction 38.7µg/mL
Ligation
LacP | pSB3C5 | Ligation High | total |
---|---|---|---|
10 | 2 | 6 | 18 |
at 4℃ for overnight
Transformation
Lacp+pSB3C5 | competent cell | total |
---|---|---|
1 | 10 | 11 |
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 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
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
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
98℃, 10sec
60℃, 30sec
68℃, 30sec
electrophoresis
1. 100bp Ladder
2. torA signal (272bp)
3. pspA (969bp)
4. 100bp Ladder
March 6
Restriction
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.
And we did gel extraction on 3/7 and get 40.0μg/mL GFP.
PCR
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
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
Kil | EcoR1 | Spe1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.3 | 0.3 | 3 | 0.3 | 16.1 | 30 |
DT | EcoR1 | Xba1 | Buffer2 | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |
at 37℃ for 2 hours
→ purification 37.7ng/μL
Ligation
Kil | pSB1C3 | Ligation High Ver.2 | total |
---|---|---|---|
4 | 2 | 3 | 9 |
- Kil : 350fmol
- pSB1C3 : 29fmol
at 16℃ for overnight
March 7
Electrophoresis
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
VectorDNA | GFP | Ligation High Ver.2 | total |
---|---|---|---|
5 | 15 | 10 | 30 |
Restriction
torA | EcoR1 | Spe1 | bufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.3 | 0.3 | 3 | 0.3 | 16.1 | 30 |
pspA | EcoR1 | Spe1 | bufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
5 | 0.3 | 0.3 | 3 | 0.3 | 21.1 | 30 |
at 37℃ for 1.5 hours
Purification
torA→31.8ng/µL
pspA→49.3ng/µL
Ligation
torA | pSB1C3 | Ligation High Ver.2 | total |
---|---|---|---|
3 | 3 | 3 | 9 |
pspA | pSB1C3 | Ligation High Ver.2 | total |
---|---|---|---|
4 | 2 | 3 | 9 |
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
pSB4K5 | EcoR1 | Spe1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
20 | 0.2 | 0.2 | 3 | 0.2 | 6.4 | 30 |
at 37℃ for 1 hour.
→Purification : 36.6ng/µL
Ligation
Kil | pSB4K5 | Ligation High Ver.2 | total |
---|---|---|---|
10 | 1 | 5 | 16 |
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
torA | pspA | competent cell | total |
---|---|---|---|
1 | 0 | 10 | 11 |
0 | 1 | 10 | 11 |
We use commercially available competent cells in this time.
March 9
Restriction
tatABCD | Xba1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.2 | 0.2 | 3 | 0.3 | 16.3 | 30 |
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 Taq | VF | VR | MilliQ | total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
94℃ 2min
94℃ 30sec
55℃ 30sec
68℃ 6sec
25cycles
Ligation
tatABCD | constP J23107 | Ligation High Ver.2 | total |
---|---|---|---|
5 | 1 | 3 | 9 |
tatABCD : 227fmol
constP J23107 : 21fmol
Transformation
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.
pSB4K5 : 80.5μg/mL
March 10
Screening PCR
Quick Taq | VF | VR | MilliQ | total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
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-pSB3C5 | Spe1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.2 | 0.2 | 2 | 0.2 | 7.4 | 20 |
for 2.5 hours at 37℃
→purification 29.0ng/μL
torA | Xba1 | Pst1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.2 | 0.2 | 2 | 0.2 | 7.4 | 20 |
for 2.5 hours at 37℃
→purification 91.8ng/μL
Ligation
torA | Lacp-pSB3C5 | Ligation High Ver.2 | total |
---|---|---|---|
4 | 2 | 3 | 9 |
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 Taq | VF | VR | MilliQ | total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
Electrophoresis
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.
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) | 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
pspA | EcoR1 | Spe1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.5 | 0.5 | 3 | 0.3 | 15.7 | 30 |
at 37℃ for 4 hours
→ We did purification and got 48.3ng/μL pspA.
Ligation
pspA | pSB1C3 | MilliQ | Ligation High Ver.2 | total |
---|---|---|---|---|
4 | 2 | 0 | 3 | 9 |
2 | 2 | 0 | 2 | 6 |
0 | 2 | 2 | 2 | 6 |
March 13
Miniprep
pSB1C3 74.2µg/mL 1.65 (260/280) 1.31 (260/230)
March 14
Restriction
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.
Ligation
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
- 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
GFP | EcoR1 | Spe1 | Buffer2 | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |
DT | EcoR1 | Xba1 | Buffer2 | BSA | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |
for 2 hours at 37℃.
Miniprep
pspA (pSB1C3) 40.5ng/µL
Ligation
pspA | DT | Ligation High Ver.2 | total |
---|---|---|---|
5 | 1.5 | 3 | 9.5 |
- pspA : 385fmol
- DT : 36fmol
Transformation
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
Quick Taq | Primer-r | Primer-f | MilliQ | total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
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 Taq | Primer-r | Primer-f | MilliQ | total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 6sec
→25cycles
Electrophoresis
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
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.
We did gel extraction for product with EcoR1, Spe1. We got 46.1ng/µL pSB1C3.
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℃.
We did this to confirm.
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℃.
We did this to confirm.
Ligation
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 overnight
- torA→767fmol
- pSB1C3→68fmol
March 17
Miniprep
J23107-tatABCD 72.7ng/µL
pspA-DT 50.5ng/µL
Checking the Insert
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.
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
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.
Ligation
Kil | DT | Ligation High Ver.2 | total |
---|---|---|---|
10 | 2 | 6 | 18 |
We did this for an hour at 16℃.
Restriction
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℃
Ligation
pspA | DT | Ligation High Ver.2 | total |
---|---|---|---|
5 | 5 | 5 | 15 |
pspA | pSB1C3 | Ligation High Ver.2 | total |
---|---|---|---|
4 | 1 | 3 | 8 |
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 Taq | Primer-R | Primer-F | MilliQ | total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
- pspA→○
- pspA-DT→○
- GFP-DT→○
- torA→×
- Kil-DT 6 of 8 sumples→○
Quick Taq | VR | VF | MilliQ | total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
- pspA→○
- pspA-DT→×
Restriction
torA | EcoR1 | Spe1 | BSA | BufferM | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.3 | 0.3 | 0.3 | 3 | 16.1 | 30 |
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.
torA 34.2ng/µL
DT 28.0ng/µL
March 21
Miniprep
GFP-DT-1 : 77.7ng/µL
GFP-DT-2 : 67.6ng/µL
Restriction
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
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.
Ligation
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.
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
98℃, 10sec
60℃, 30sec
68℃, 10sec
→30cycles
→Purification 110.7ng/µL
Restriction
torA | EcoR1 | Spe1 | BSA | BufferM | MilliQ | total |
---|---|---|---|---|---|---|
10 | 0.2 | 0.2 | 0.3 | 3 | 16.3 | 30 |
Ligation
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
- 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 Taq | VF | VR | MilliQ | total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
E.coli in liquid culture that had pspA(pSB1C3) also expressed RFP.
Restriction
pspA | EcoR1 | Spe1 | BufferM | BSA | MilliQ | total |
---|---|---|---|---|---|---|
5 | 0.3 | 0.3 | 3 | 0.3 | 21.1 | 30 |
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+pSB3C5-8 | Spe1 | Pst1 | Buffer2 | BSA | MilliQ | total |
---|---|---|---|---|---|---|
20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |
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
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) | 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 |
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]
Ligation
Lacp (pSB3C5) | torA-GFP-DT | Ligation High Ver.2 | total |
---|---|---|---|
1 | 5 | 3 | 9 |
- Lacp : 22fmol
- torA-GFP-DT : 197fmol
pspA | pSB1C3 | Ligation High Ver.2 | total |
---|---|---|---|
10 | 1 | 5 | 16 |
pspA | DT | Ligation High Ver.2 | total |
---|---|---|---|
10 | 1 | 5 | 16 |
- pspA : 180fmol
- pSB1C3 : 18fmol
- DT : 16fmol
LacP(pSB3C5) | Kil-DT | Ligation High Ver.2 | total |
---|---|---|---|
1 | 5 | 3 | 9 |
for 2 hours at 16℃
Transformation
Lacp-Kil-DT | competent cell | total |
---|---|---|
1 | 10 | 11 |
March 27
Miniprep
We retryed miniprep of torA(pSB1C3).
We got torA and its concentration was 39.3[ng/µL].
Transformation
Name | Well | Sample | Competent Cells | Total | Plate | Colony |
---|---|---|---|---|---|---|
BBa_K117004 | 14J(2011 plate2) | 5 | 20 | ? | ? | ? |
We added 100[µL] of culture medium before we started culturing the E.coli.
Screening PCR
Quick Taq | VF2 | VR | MilliQ | Total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
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 30
Ligation
kil+DT(XbaI,PstI) | LacP(SpeI,PstI) | Ligation High | Total |
---|---|---|---|
30 | 6 | 36 | 72 |
July 31
Restriction Enzyme Processing
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
Restriction Enzyme Processing
lacP+kil+DT | BSA | 10×H Buffer | EcoRI | PstI | MilliQ | Total |
---|---|---|---|---|---|---|
5.0 | 0.5 | 3.0 | 0.5 | 0.5 | 20.5 | 30.0 |
lacP middle copy | BSA | 10×H Buffer | EcoRI | PstI | MilliQ | Total |
---|---|---|---|---|---|---|
10.0 | 0.5 | 3.0 | 0.5 | 0.5 | 15.5 | 30.0 |
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
pSB4K5 | BSA | 10×H Buffer | EcoRI | PstI | MilliQ | Total |
---|---|---|---|---|---|---|
8.0 | 0.5 | 3.0 | 0.5 | 0.5 | 17.5 | 30.0 |
B0034 | BSA | 10×H Buffer | SpeI | PstI | MilliQ | Total |
---|---|---|---|---|---|---|
12.0 | 0.5 | 3.0 | 0.5 | 0.5 | 13.5 | 30.0 |
DNA purification
Ligation
lacP+kil+DT | pSB4K5 | ligation high | Total |
---|---|---|---|
15 | 15 | 15 | 45 |
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 SpeI PstI | GFP+DT XbaI PstI | ligation high | Total |
---|---|---|---|
2 | 3 | 4 | 9 |
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 Taq | VF | VF | MilliQ | Total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
pspA
10×Buffer for KOD-Plus-Ver2 | 2mM dNTPs | 25mM MgSO4 | Primer PsPA f-p | Primer PsPA r-s | KOD-Plus- | MilliQ | Total |
---|---|---|---|---|---|---|---|
5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |
Electrophoresis
写真貼ってください
positive control(GFP+DT)
negative control(MilliQ)
① ~⑥ colony PCR lacP+kil+DT
次の写真
① 1000kb 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 1000kb ladder
August 15
Colony PCR
pspA
2×Quick Taq | pspA r-s | pspA f-p | MilliQ | Total |
---|---|---|---|---|
25 | 1 | 1 | 23 | 50 |
94℃, 2min
94℃, 30sec
56℃, 30sec
68℃, 1min
→35cycles
August 16
August 17
Colony PCR
pspA
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 |
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
94℃, 15sec
55℃, 30sec
68℃, 1min
→35cycles
Electrophoresis
写真貼ってください