Team:Kyoto/Florigen/Note

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 +
= Florigen Notebook =
 +
<div class="_kyoto-note">
 +
==August 2==
 +
'''Mutation of FT'''  <small>by Sato</small><br>
 +
FT gene has two BioBrick restriction enzyme sites, EcoR1 and Pst1 which is next to each other.<br>
 +
So we tried to delete both at once by using two primers with mutation.<br>
 +
{|class="wikitable"
 +
|+Inverse PCR
 +
!10xBufer!!2mM dNTPs!!primer fwd!!primer rev!!template!!polymerase!!MilliQ!!Total
 +
|-
 +
|5||5||1.5||1.5||0.5||1||35.5||50
 +
|}
 +
94℃ 2min, (98℃ 10sec, 68℃ 4min)x4cycles, 4℃ Hold
 +
 +
{|class="wikitable"
 +
|+Dpn1 Digestion
 +
!PCR product!!Dpn1
 +
|-
 +
|50||2
 +
|}  37℃,1h incubate
 +
 +
{|class="wikitable"
 +
|+Self-ligation
 +
!product!!MilliQ!!Ligase!!T4 Kinase!!Total
 +
|-
 +
|2||7||5||1||15
 +
|}
 +
16℃, 1h incubate
 +
 +
{|class="wikitable"
 +
|+Transformation
 +
!competent cell!!DNA
 +
|-
 +
|20||2
 +
|}
 +
Cells were stored on ice for 30min. <br>
 +
After 42℃ 60sec heat shock, cells were stored on ice for 2min.<br>
 +
Then cells were precultureed at 37℃ for 1hr, plated to Kanamycin plate.
 +
<br>
 +
<br>
 +
 +
==August 13==
 +
'''Liquid culture'''<br>
 +
FT at 37°C, for overnight.
 +
 +
==August 14==
 +
'''Miniprep of FT'''<small> : by Sato, Takeuchi</small><br>
 +
The concentration was 81.5ng/uL<br><br>
 +
'''Restriction digestion and Electrophoresis'''<small> : by Sato</small><br>
 +
To check wheter mutation was succeed, we did restriction enzyme digestion.
 +
{|class="wikitable"
 +
!DNA(FT,80ng/uL)!!10xBuferH!!EcoR1!!Pst1!!MilliQ!!Total
 +
|-
 +
|5||2||1||-||12||20
 +
|-
 +
|5||2||-||1||12||20
 +
|}
 +
37°C 2h incubate<br>
 +
We did electrophoresis of three FT plasmids, non-cutted, EcoR1 cutted, Pst1 cutted.<br>
 +
However, we couldn't get any bands (data not shown.)<br>
 +
<br>
 +
'''Liquid culture'''<br>
 +
FT (4mL)
 +
 +
==August 15==
 +
'''Miniprep of FT'''<small> : by Sato, Takeuchi, Hyungcheol</small><br>
 +
We couldn't get enough concentration of plasmids.<br><br>
 +
'''Electrophoresis'''<small> : by Sato</small><br>
 +
We retried electrophoresis of three samples same as yesterday.<br>
 +
However, we couldn't get any bands as well.
 +
 +
==August 16==
 +
'''Transformation'''<small> : by Takeuchi, Ota</small><br>
 +
{| class="wikitable"
 +
!Name||Well||Sample||Competent Cells||Total||Plate||Colony
 +
|-
 +
|FT||-||1 &micro;L||10||11||LB (Kan+)||&#xD7;
 +
|-
 +
|pSB1C3||1-3-A||1||10||11||LB (CP+)||&#x25CB;
 +
|-
 +
|I719005||1-15-N||1||10||11||LB (Amp+)||&#x25CB;
 +
|}
 +
==August 17==
 +
'''Transformation'''<small> : by Takeuchi</small><br>
 +
{| class="wikitable"
 +
!Name||Well||Sample||Competent Cells||MilliQ||Total||Plate||Colony
 +
|-
 +
|FT||-||2||2||18||22||LB (Kan+)||&#xD7;
 +
|}
 +
We found that mutation of FT was not successful.
 +
==August 20==
 +
We decided to do PCR using FT specific primers before mutation.<br>
 +
'''PCR of FT''' <small> : by Sato</small><br>
 +
{|class="wikitable"
 +
!10xBufer!!dNTPs!!MgSO4!!primer fwd!!primer rev!!template!!polymerase!!MilliQ!!Total
 +
|-
 +
|5||5||3||1||1||1(130ng/&micro;L)||1(KOD plus neo)||33||50
 +
|}
 +
94°C 2min,  (98°C 10sec, 68°C 15sec)x30cycles,  4°C Hold<br>
 +
After the ethanol precipitation, we diluted in 30&micro;L of MilliQ.<br>
 +
The concentration was 203ng/&micro;L.
 +
<br>
 +
<br>
 +
'''Electrophoresis'''[[File:Electrophoresis0821.png|400px|thumb|right]]
 +
{| class="wikitable"
 +
!Lane!!Name!!length(bp)
 +
|-
 +
|1||1kb ladder||-
 +
|-
 +
|2||FT||600
 +
|}
 +
<br>
 +
==August 21==
 +
'''Restriction digestion''' <small> : by Sato</small><br>
 +
{|class="wikitable"
 +
!DNA(FT,203ng/&micro;L)!!10xBuferM!!Xba11!!Pst1!!MilliQ!!Total
 +
|-
 +
|10||4||1||1||24||40
 +
|}
 +
37°C, 5h incubate<br>
 +
After the ethanol precipitation, we diluted in 30&micro;L of MilliQ.<br>
 +
The concentration was 54,9ng/&micro;L.<br>
 +
<br>
 +
'''Ligation''' <small> : by Sato</small><br>
 +
{| class="wikitable"
 +
!colspan="2"|Vector||colspan="2"|Insert||Ligation High Ver.2
 +
|-
 +
|pSB1C3||1||FT||10||5.5
 +
|}
 +
 +
'''Liquid culture'''<br>
 +
T7 promoter, pSB1C3 (4mL)
 +
<br>
 +
<br>
 +
==August 22==
 +
'''Miniprep'''<small> : by Sato</small><br>
 +
{| class="wikitable"
 +
!T7 promoter||pSB1C3
 +
|-
 +
|85.3ng/&micro;L||82.93ng/&micro;L
 +
|}
 +
<br>
 +
 +
==August 23==
 +
'''Ethanol Precipitation<br>'''
 +
<small>diluted in 20µL 79.3ng/µL</small><br>
 +
<br>mada dekite nai
 +
 +
 +
==August 24==
 +
'''Restriction enzyme processing'''<br>
 +
{|class="wikitable"
 +
!T7 promoter(85.3ng/µL)!!Spel!!Pstl!!buffer M!!MiliQ!!Total
 +
|-
 +
|10||1||1||2||6||20
 +
|}
 +
->purifying column 33.4ng/µL(dissolution 40µL)<br>
 +
{|class="wikitable"
 +
!pSB1C3(82.9ng/µL)!!Xbal!!Spel!!buffer M!!MiliQ!!Total
 +
|-
 +
|20||1||1||4||14||40
 +
|}
 +
->gene clean2 39.9ng/µL(dissolution 40µL)<br>
 +
<br>
 +
<<picture1,2>>
 +
<br>
 +
'''Ligation'''<br>
 +
{|class="wikitable"
 +
!FT(600bp, 79.3ng/µL)!!pSB1C3(2000bp,39.9ng/µL)!!Ligation High Ver.2
 +
|-
 +
|3µL => 597fmol||2µL => 60fmol||2.5µL
 +
|}
 +
{|class="wikitable"
 +
!FT(600bp, 79.3ng/µL)!!T7(2100bp,33.4ng/µL)!!Ligation High Ver.2
 +
|-
 +
|2.4µL => 478fmol||2µL => 48fmol||2.2µL
 +
|}
 +
=> 16℃,1hr incubate
 +
 +
==August 27==
 +
'''Colony PCR'''<br>
 +
{|class="wikitable"
 +
!2X Quick Tag!!VF2!!VR!!MiliQ!!Total
 +
|-
 +
|25||1||1||23||50
 +
|}
 +
Lane1: 1kb ladder<br>
 +
Lane2~16: FT(pSB1C3) about 800bp<br>
 +
 +
'''FT(TOPO) PCR(re)'''<br>
 +
 +
{|class="wikitable"
 +
!buffer!!dNTPs!!MgSO4!!primer f!!primer r!!Template(130ng/µL)!!KODplus neo!!MilliQ!!Total
 +
|-
 +
|5||5||3||1||1||1||1||33||50
 +
|}
 +
 +
{| class="wikitable"
 +
!94℃||98℃||68℃||cycles
 +
|-
 +
|2min||10sec||15sec||30
 +
|}
 +
<br>
 +
'''Liquid culture'''    <small>by Nobeyama</small><br>
 +
FT 4ml<br>
 +
 +
==August 28==
 +
 +
'''Mutation of FT (re)'''<br><br>
 +
inverse PCR<br>
 +
{|class="wikitable"
 +
!MilliQ!!buffer!!dNTP!!primer f!!primer r!!FT(130ng/µL)!!KODplus!!Total
 +
|-
 +
|35.5||5||5||1.5||1.5||0.5||1||50
 +
|}
 +
{| class="wikitable"
 +
!94℃||98℃||68℃||cycles
 +
|-
 +
|2min||10sec||4min||18
 +
|}<br>
 +
Lane1: 1kb ladder<br>
 +
Lane2: FT<br>
 +
 +
'''Miniprep FT(TOPO''')<br>
 +
158ng/µL<br><br>
 +
'''Tranformation'''<br>
 +
competent cell: 20<br>
 +
BBa.I746902  : 2<br>
 +
(plate 316f)<br>
 +
(GFP generator of pBAD/araC-mut3 GFP:6His-DT)<br>
 +
 +
==August 29==
 +
'''Mutaion of FT(re;re)'''<br><br>
 +
'''Inverse PCR'''<br><br>
 +
first
 +
{|class="wikitable"
 +
!MilliQ!!buffer!!dNTP!!primer f!!primer r!!FT(130ng/µL)!!KODplus!!Total
 +
|-
 +
|35.5||5||5||1.5||1.5||0.5||1||50
 +
|}
 +
second
 +
{|class="wikitable"
 +
!MilliQ!!buffer!!dNTP!!primer f!!primer r!!FT(52ng/µL)!!KODplus!!Total
 +
|-
 +
|35||5||5||1.5||1.5||1||1||50
 +
|}
 +
{| class="wikitable"
 +
!94℃||98℃||68℃||cycles
 +
|-
 +
|2min||10sec||4min||30
 +
|}<br>
 +
Lane1: 1kb ladder<br>
 +
Lane2: first Template 130ng/µL<br>
 +
Lane3: second Template 53ng/µL<br>
 +
 +
{| class="wikitable"
 +
!first sample||Dpnl||total
 +
|-
 +
|45µL||2µL||47µL
 +
|}
 +
in 37℃, 1 hour<br>
 +
 +
'''Self-Ligation'''<br>
 +
 +
{| class="wikitable"
 +
!PCR products||MilliQ||Ligation High||T4 kinase||total
 +
|-
 +
|2µL||7µL||5µL||1µL||15µL
 +
|}<br>
 +
in 16℃, 1.5hour incubate<br>
 +
Transformation<br>
 +
competent cell: 20<br>
 +
DNA          : 2<br><br>
 +
Liquid culture(I746902): 3mL
 +
 +
==August 30==
 +
'''Liquid culture(FT) 4mL x2'''<br>
 +
 +
==August 31==
 +
'''Miniprep(FT)'''<br>
 +
 
 +
(1) 64.9ng/µL<br>
 +
(2) 52.6ng/µL<br>
 +
 +
'''Restriction enzyme processing (Mutation check)'''<br>
 +
{| class="wikitable"
 +
!FT(52.6ng/µL)||bufferH||E.coli||Pst1||MilliQ||total
 +
|-
 +
|1µL||5µL||0.5µL||0.5µL||3.5µL||10µL
 +
|}<br>
 +
in 37℃,1.5hour<br>
 +
'''PCR(RBS primer)'''<br>
 +
 +
{|class="wikitable"
 +
!buffer for KODplus neo!!dNTPs!!MgSO4!!primer f!!primer r!!Template(52.6ng/µL)!!KODplus neo!!MilliQ!!Total
 +
|-
 +
|5||5||3||1||1||1||1||33||50
 +
|}<br>
 +
 +
{| class="wikitable"
 +
!94℃||98℃||68℃||cycles
 +
|-
 +
|2min||10sec||15sec||30
 +
|}<br>
 +
 +
Lane1: 1kb ladder<br>
 +
Lane2: FT<br>
 +
Lane3: FT(E.coli)<br>
 +
Lane4: FT(Pst1)<br>
 +
Lane5: FT PCR<br><br>
 +
 +
'''PCR(re)'''<br>
 +
 +
{|class="wikitable"
 +
!buffer!!dNTPs!!MgSO4!!primer f!!primer r!!Template!!KOD neo!!MilliQ!!Total
 +
|-
 +
|5||5||3||1||1||1||1||33||50
 +
|}<br>
 +
{| class="wikitable"
 +
!94℃||98℃||68℃||cycles
 +
|-
 +
|2min||10sec||15sec||35
 +
|}<br>
 +
 +
Lane1: 1kb ladder<br>
 +
Lane2: FT<br>
 +
 +
'''PCR(re)'''<br>
 +
 +
{|class="wikitable"
 +
!buffer!!dNTPs!!MgSO4!!primer f!!primer r!!Template!!KOD neo!!MilliQ!!Total
 +
|-
 +
|5||5||2||1||1||1||1||34||50
 +
|}<br>
 +
{| class="wikitable"
 +
!94℃||98℃||68℃||cycles
 +
|-
 +
|2min||10sec||10sec||30
 +
|}<br>
 +
 +
==September 2==
 +
 +
'''PCR(re;re)'''<br><br>
 +
 +
first
 +
{|class="wikitable"
 +
!buffer!!dNTPs!!MgSO4!!primer f!!primer r!!Template(1ng/µL)!!KODplus neo!!MilliQ!!Total
 +
|-
 +
|5||5||3||1||1||1||1||33||50
 +
|}<br>
 +
 +
second
 +
{|class="wikitable"
 +
!buffer!!dNTPs!!MgSO4!!primer f!!primer r!!Template(10ng/µL)!!KODplus neo!!MilliQ!!Total
 +
|-
 +
|5||5||3||1||1||1||1||33||50
 +
|}<br>
 +
{| class="wikitable"
 +
!94℃||98℃||68℃||cycles
 +
|-
 +
|2min||10sec||10sec||25
 +
|}<br>
 +
 +
Lane1: first Template 1ng<br>
 +
Lane2: second Template 10ng<br>
 +
Lane3: 1kb ladder<br>
 +
 +
refine first Template => 132ng/µL<br>
 +
</div>
{{Kyoto/footer}}
{{Kyoto/footer}}

Latest revision as of 05:19, 7 September 2012

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Contents

Florigen Notebook

August 2

Mutation of FT by Sato
FT gene has two BioBrick restriction enzyme sites, EcoR1 and Pst1 which is next to each other.
So we tried to delete both at once by using two primers with mutation.

Inverse PCR
10xBufer2mM dNTPsprimer fwdprimer revtemplatepolymeraseMilliQTotal
551.51.50.5135.550

94℃ 2min, (98℃ 10sec, 68℃ 4min)x4cycles, 4℃ Hold

Dpn1 Digestion
PCR productDpn1
502
37℃,1h incubate
Self-ligation
productMilliQLigaseT4 KinaseTotal
275115

16℃, 1h incubate

Transformation
competent cellDNA
202

Cells were stored on ice for 30min.
After 42℃ 60sec heat shock, cells were stored on ice for 2min.
Then cells were precultureed at 37℃ for 1hr, plated to Kanamycin plate.

August 13

Liquid culture
FT at 37°C, for overnight.

August 14

Miniprep of FT : by Sato, Takeuchi
The concentration was 81.5ng/uL

Restriction digestion and Electrophoresis : by Sato
To check wheter mutation was succeed, we did restriction enzyme digestion.

DNA(FT,80ng/uL)10xBuferHEcoR1Pst1MilliQTotal
521-1220
52-11220

37°C 2h incubate
We did electrophoresis of three FT plasmids, non-cutted, EcoR1 cutted, Pst1 cutted.
However, we couldn't get any bands (data not shown.)

Liquid culture
FT (4mL)

August 15

Miniprep of FT : by Sato, Takeuchi, Hyungcheol
We couldn't get enough concentration of plasmids.

Electrophoresis : by Sato
We retried electrophoresis of three samples same as yesterday.
However, we couldn't get any bands as well.

August 16

Transformation : by Takeuchi, Ota

NameWellSampleCompetent CellsTotalPlateColony
FT-1 µL1011LB (Kan+)×
pSB1C31-3-A11011LB (CP+)○
I7190051-15-N11011LB (Amp+)○

August 17

Transformation : by Takeuchi

NameWellSampleCompetent CellsMilliQTotalPlateColony
FT-221822LB (Kan+)×

We found that mutation of FT was not successful.

August 20

We decided to do PCR using FT specific primers before mutation.
PCR of FT  : by Sato

10xBuferdNTPsMgSO4primer fwdprimer revtemplatepolymeraseMilliQTotal
553111(130ng/µL)1(KOD plus neo)3350

94°C 2min, (98°C 10sec, 68°C 15sec)x30cycles, 4°C Hold
After the ethanol precipitation, we diluted in 30µL of MilliQ.
The concentration was 203ng/µL.

Electrophoresis
Electrophoresis0821.png
LaneNamelength(bp)
11kb ladder-
2FT600


August 21

Restriction digestion  : by Sato

DNA(FT,203ng/µL)10xBuferMXba11Pst1MilliQTotal
104112440

37°C, 5h incubate
After the ethanol precipitation, we diluted in 30µL of MilliQ.
The concentration was 54,9ng/µL.

Ligation  : by Sato

VectorInsertLigation High Ver.2
pSB1C31FT105.5

Liquid culture
T7 promoter, pSB1C3 (4mL)

August 22

Miniprep : by Sato

T7 promoterpSB1C3
85.3ng/µL82.93ng/µL


August 23

Ethanol Precipitation
 diluted in 20µL 79.3ng/µL

mada dekite nai


August 24

Restriction enzyme processing

T7 promoter(85.3ng/µL)SpelPstlbuffer MMiliQTotal
10112620

->purifying column 33.4ng/µL(dissolution 40µL)

pSB1C3(82.9ng/µL)XbalSpelbuffer MMiliQTotal
201141440

->gene clean2 39.9ng/µL(dissolution 40µL)

<<picture1,2>>
Ligation

FT(600bp, 79.3ng/µL)pSB1C3(2000bp,39.9ng/µL)Ligation High Ver.2
3µL => 597fmol2µL => 60fmol2.5µL
FT(600bp, 79.3ng/µL)T7(2100bp,33.4ng/µL)Ligation High Ver.2
2.4µL => 478fmol2µL => 48fmol2.2µL

=> 16℃,1hr incubate

August 27

Colony PCR

2X Quick TagVF2VRMiliQTotal
25112350

Lane1: 1kb ladder
Lane2~16: FT(pSB1C3) about 800bp

FT(TOPO) PCR(re)

bufferdNTPsMgSO4primer fprimer rTemplate(130ng/µL)KODplus neoMilliQTotal
55311113350
94℃98℃68℃cycles
2min10sec15sec30


Liquid culture by Nobeyama
FT 4ml

August 28

Mutation of FT (re)

inverse PCR

MilliQbufferdNTPprimer fprimer rFT(130ng/µL)KODplusTotal
35.5551.51.50.5150
94℃98℃68℃cycles
2min10sec4min18

Lane1: 1kb ladder
Lane2: FT

Miniprep FT(TOPO)
158ng/µL

Tranformation
competent cell: 20
BBa.I746902  : 2
(plate 316f)
(GFP generator of pBAD/araC-mut3 GFP:6His-DT)

August 29

Mutaion of FT(re;re)

Inverse PCR

first

MilliQbufferdNTPprimer fprimer rFT(130ng/µL)KODplusTotal
35.5551.51.50.5150

second

MilliQbufferdNTPprimer fprimer rFT(52ng/µL)KODplusTotal
35551.51.51150
94℃98℃68℃cycles
2min10sec4min30

Lane1: 1kb ladder
Lane2: first Template 130ng/µL
Lane3: second Template 53ng/µL

first sampleDpnltotal
45µL2µL47µL

in 37℃, 1 hour

Self-Ligation

PCR productsMilliQLigation HighT4 kinasetotal
2µL7µL5µL1µL15µL

in 16℃, 1.5hour incubate
Transformation
competent cell: 20
DNA  : 2

Liquid culture(I746902): 3mL

August 30

Liquid culture(FT) 4mL x2

August 31

Miniprep(FT)

(1) 64.9ng/µL
(2) 52.6ng/µL

Restriction enzyme processing (Mutation check)

FT(52.6ng/µL)bufferHE.coliPst1MilliQtotal
1µL5µL0.5µL0.5µL3.5µL10µL

in 37℃,1.5hour
PCR(RBS primer)

buffer for KODplus neodNTPsMgSO4primer fprimer rTemplate(52.6ng/µL)KODplus neoMilliQTotal
55311113350

94℃98℃68℃cycles
2min10sec15sec30

Lane1: 1kb ladder
Lane2: FT
Lane3: FT(E.coli)
Lane4: FT(Pst1)
Lane5: FT PCR

PCR(re)

bufferdNTPsMgSO4primer fprimer rTemplateKOD neoMilliQTotal
55311113350

94℃98℃68℃cycles
2min10sec15sec35

Lane1: 1kb ladder
Lane2: FT

PCR(re)

bufferdNTPsMgSO4primer fprimer rTemplateKOD neoMilliQTotal
55211113450

94℃98℃68℃cycles
2min10sec10sec30

September 2

PCR(re;re)

first

bufferdNTPsMgSO4primer fprimer rTemplate(1ng/µL)KODplus neoMilliQTotal
55311113350

second

bufferdNTPsMgSO4primer fprimer rTemplate(10ng/µL)KODplus neoMilliQTotal
55311113350

94℃98℃68℃cycles
2min10sec10sec25

Lane1: first Template 1ng
Lane2: second Template 10ng
Lane3: 1kb ladder

refine first Template => 132ng/µL

Retrieved from "http://2012.igem.org/Team:Kyoto/Florigen/Note"