Template:Kyoto/Notebook/Sep 4

From 2013.igem.org

(Difference between revisions)
(Colony PCR)
(Sep 4)
Line 1: Line 1:
==Sep 4==
==Sep 4==
 +
===Ligation===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
 +
|-
 +
|experiment||9/3 DT(EcoRI&XbaI)||3.2&micro;L||9/3 RBS-lysis1||2.4&micro;L||2.7&micro;L
 +
|-
 +
|experiment||9/3 DT(EcoRI&XbaI) ||3.0&micro;L ||9/3 RBS-lysis2||4.7&micro;L ||4.0&micro;L
 +
|-
 +
|experiment||9/3 DT(EcoRI&XbaI) ||3.0&micro;L ||9/3 RBS-lysis3||8.8&micro;L ||4.0&micro;L
 +
|-
 +
|experiment||9/3 DT(EcoRI&XbaI) ||3.0&micro;L ||9/3 aptamer12-1R(EcoRI&SpeI) ||1.9&micro;L ||2.45&micro;L
 +
|-
 +
|experiment||9/2 pSB1C3(XbaI&PstI)||10.6&micro;L ||9/3 pT181attenuator (XbaI& PstI)||3.2&micro;L ||4.0&micro;L
 +
|-
 +
|experiment||9/2 pSB1C3(XbaI&PstI)||9.6&micro;L ||9/3 pT181antisense (XbaI& PstI)||2.1&micro;L ||4.0&micro;L
 +
|-
 +
|experiment||9/2 pSB1C3(EcoRI&SpeI)||16.7&micro;L ||9/3 aptamer12-1R(EcoRI&SpeI) ||2.0&micro;L ||4.0&micro;L
 +
|}
 +
incubate 16 &deg;C 1 hour
 +
</div>
 +
 +
 +
 +
 +
 +
 +
 +
===Transformation===
 +
<div class="experiment">
 +
<span class="author">Tatsui</span>
 +
{| class="wikitable"
 +
!Name||Sample||Competent Cells||Plate
 +
|-
 +
|9/4 RBS-lysis1-DT ||2&micro;L||20&micro;L||Amp
 +
|-
 +
|9/4 RBS-lysis2-DT ||2&micro;L||20&micro;L||Amp
 +
|-
 +
|9/4 RBS-lysis3-DT ||2&micro;L||20&micro;L||Amp
 +
 +
|9/4 aptamer12-1R-DT||2&micro;L||20&micro;L||Amp
 +
|-
 +
|9/4 pT181attenuator –pSB1C3||2&micro;L||20&micro;L||Amp
 +
|-
 +
|9/4 pT181antisense-pSB1C3 ||2&micro;L||20&micro;L||Amp
 +
|-
 +
|9/4 aptamer12-1R –pSB1C3||2&micro;L||20&micro;L||Amp
 +
|-
 +
|9/4 pSB4K5(2013plate5 56) ||2&micro;L||20&micro;L||Amp
 +
|-
 +
|9/3 Mutagenesis product 1(KaiABC) ||2&micro;L||20&micro;L||Amp
 +
|-
 +
|9/3 Mutagenesis product 2(NC) ||2&micro;L||20&micro;L||Amp
 +
|}
 +
</div>
 +
 +
===Miniprep===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{|class="wikitable"
 +
!DNA||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|DT||193.1||1.93||1.81
 +
|-
 +
|Spinach-DT||373.3||1.92||1.69
 +
|-
 +
|RBS-lysis1||332.0||1.95||1.62
 +
 +
|}
 +
===Master Plate===
 +
<!-- ここから -->
 +
<div class="experiment">
 +
<span class="author">Hirano</span>
 +
{| class="wikitable"
 +
!Number||Use LB plate (+CP)
 +
|-
 +
|1||8/10 Plac(CP)-2
 +
|-
 +
|2|| 8/10 Plac(CP)-2
 +
|-
 +
|3||8/10 Plac(CP)-2
 +
|}
 +
</div>
 +
===Master Plate===
 +
<!-- ここから -->
 +
<div class="experiment">
 +
<span class="author">Hirano</span>
 +
{| class="wikitable"
 +
!Number||Use LB plate (+Amp)
 +
|-
 +
|1||8/9 J23100(Amp)-1
 +
|-
 +
|2||8/9 J23100(Amp)-1
 +
|-
 +
|3||8/9 J23100(Amp)-1
 +
|}
 +
</div>
 +
 +
 +
===Liquid Culture===
 +
<div class="experiment">
 +
<span class="author">Hirano</span>
 +
{| class="wikitable"
 +
!Sample||medium
 +
|-
 +
|8/10 Plac(CP)-2||Plusgrow medium(+CP)
 +
|-
 +
|8/9 J23100(Amp)-1||Plusgrow medium(+Amp)
 +
|}
 +
</div>
 +
 +
===PCR===
 +
<!-- ここから -->
 +
<div class="experiment">
 +
<span class="author">Hirano</span>
 +
{| class="wikitable"
 +
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||F primer (RBS-luxI-DT-cloning-fond) || R primer(RBS-luxI-DT-cloning-rex)||MilliQ||total
 +
|-
 +
|8/29 p&lambda;-RBS-luxI-DT||0.3&micro;L||0.5&micro;L ||2.5&micro;L ||2.5&micro;L ||1.5&micro;L ||0.75&micro;L ||0.75&micro;L ||16.2&micro;L ||25&micro;L
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
| 94&deg;C|| 98&deg;C|| 65&deg;C|| 68&deg;C||--
 +
|-
 +
|5min||10s ||30s ||3min10s ||30cycles
 +
|}
 +
</div>
 +
<!-- ここまでをコピーしてね -->
 +
 +
===PCR===
 +
<!-- ここから -->
 +
<div class="experiment">
 +
<span class="author">Hirano</span>
 +
{| class="wikitable"
 +
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||F primer (Pbad/araC-pSB1C3-cloning-fond) ||R primer(Pbad/araC-cloning-rex)||MilliQ||total
 +
|-
 +
|8/31 Pbad/araC-RBS-RFP(1)||0.3&micro;L||0.5&micro;L ||2.5&micro;L ||2.5&micro;L ||1.5&micro;L ||0.75&micro;L ||0.75&micro;L ||16.2&micro;L ||25&micro;L
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
| 94&deg;C|| 98&deg;C|| 65&deg;C|| 68&deg;C||--
 +
|-
 +
|5min||10s ||30s ||3min10s ||30cycles
 +
|}
 +
</div>
 +
<!-- ここまでをコピーしてね -->
 +
 +
===Restriction Enzyme Digestion===
 +
<!-- こっから -->
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
! ||8/21 Pconst||SpeI||PstI||Buffer||BSA||MilliQ||total
 +
|-
 +
|2 cuts||10.5&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||0.3&micro;L||
 +
14.2&micro;L||30 &micro;L
 +
|-
 +
|NC||0.5&mic6/8ro;L||0&micro;L||0&micro;||1.0&micro;L||0.1&micro;L|| 8.4&micro;L||10 &micro;L
 +
|}
 +
 +
{| class="wikitable"
 +
! ||8/29 Plac||SpeI||PstI||Buffer||BSA||MilliQ||total
 +
|-
 +
|2 cuts||13.0&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||0.3&micro;L||
 +
11.7&micro;L||30 &micro;L
 +
|-
 +
|NC||0.7&mic6/8ro;L||0&micro;L||0&micro;||1.0&micro;L||0.1&micro;L|| 8.2&micro;L||10 &micro;L
 +
|}
 +
 +
{| class="wikitable"
 +
! ||8/20 Ptet||SpeI||PstI||Buffer||BSA||MilliQ||total
 +
|-
 +
|2 cuts||9.0&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||0.3&micro;L||
 +
15.7&micro;L||30 &micro;L
 +
|-
 +
|NC||0.5&mic6/8ro;L||0&micro;L||0&micro;||1.0&micro;L||0.1&micro;L|| 8.4&micro;L||10 &micro;L
 +
|}
 +
 +
{| class="wikitable"
 +
! ||8/20 RBS-tetR-DT||XbaI||PstI||Buffer||BSA||MilliQ||total
 +
|-
 +
|2 cuts||8.5&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||0.3&micro;L||
 +
16.2&micro;L||30 &micro;L
 +
|-
 +
|NC||0.5&mic6/8ro;L||0&micro;L||0&micro;||1.0&micro;L||0.1&micro;L|| 8.4&micro;L||10 &micro;L
 +
|}
 +
 +
{| class="wikitable"
 +
! ||spinach -DT||XbaI||PstI||Buffer||BSA||MilliQ||total
 +
|-
 +
|2 cuts||5.4&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||3.0&micro;L||
 +
16.6&micro;L||30 &micro;L
 +
|-
 +
|NC||0.3&mic6/8ro;L||0&micro;L||0&micro;||1.0&micro;L||1.0&micro;L|| 7.7&micro;L||10 &micro;L
 +
|}
 +
 +
{| class="wikitable"
 +
! ||8/17 RBS-lacZ&alpha;-DT ||XbaI||PstI||Buffer||BSA||MilliQ||total
 +
|-
 +
|2 cuts||11&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||3.0&micro;L||
 +
11&micro;L||30 &micro;L
 +
|-
 +
|NC||0.5&mic6/8ro;L||0&micro;L||0&micro;||1.0&micro;L||1.0&micro;L|| 7.5&micro;L||10 &micro;L
 +
|}
 +
</div>
 +
 +
===Transformation===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Name||Sample||Competent Cells||Total||Plate
 +
|-
 +
|Ptrc KaiC||1&micro;L||10&micro;L||11&micro;L||Amp
 +
|-
 +
|Ptrc KaiB||1&micro;L||10&micro;L||11&micro;L||Amp
 +
|-
 +
|pS&omega; ||1&micro;L||10&micro;L||11&micro;L||Amp
 +
|}
 +
</div>
 +
 +
===Electrophoresis===
 +
<div class="experiment">
 +
<span class="author">No name</span><br>
 +
1
 +
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme1||Enzyme2
 +
|-
 +
|1||100bp ladder||-||--
 +
|-
 +
|2||Pconst||SpeI||PstI
 +
|-
 +
|3||Pconst NC||--||--
 +
|-
 +
|4||Plac||SpeI||PstI
 +
|-
 +
|5||Plac NC||--||--
 +
|-
 +
|6||100bp ladder||--||--
 +
|}
 +
[[File:igku_xxxxxx.xxx]]<br>
 +
2
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme1||Enzyme2
 +
|-
 +
|1||100bp ladder||-||--
 +
|-
 +
|2||Ptet||SpeI||PstI
 +
|-
 +
|3||Ptet NC||--||--
 +
|-
 +
|4||RBS-tetR-DT||XbaI||PstI
 +
|-
 +
|5||RBS-tetR-DT NC||--||--
 +
|-
 +
|6||100bp ladder||--||--
 +
|}
 +
[[File:igku_xxxxxx.xxx]]<br>
 +
</div>
===Gel Extraction===
===Gel Extraction===

Revision as of 13:00, 24 September 2013

Contents

Sep 4

Ligation

No name

stateVectorInserterLigation High ver.2
experiment9/3 DT(EcoRI&XbaI)3.2µL9/3 RBS-lysis12.4µL2.7µL
experiment9/3 DT(EcoRI&XbaI) 3.0µL 9/3 RBS-lysis24.7µL 4.0µL
experiment9/3 DT(EcoRI&XbaI) 3.0µL 9/3 RBS-lysis38.8µL 4.0µL
experiment9/3 DT(EcoRI&XbaI) 3.0µL 9/3 aptamer12-1R(EcoRI&SpeI) 1.9µL 2.45µL
experiment9/2 pSB1C3(XbaI&PstI)10.6µL 9/3 pT181attenuator (XbaI& PstI)3.2µL 4.0µL
experiment9/2 pSB1C3(XbaI&PstI)9.6µL 9/3 pT181antisense (XbaI& PstI)2.1µL 4.0µL
experiment9/2 pSB1C3(EcoRI&SpeI)16.7µL 9/3 aptamer12-1R(EcoRI&SpeI) 2.0µL 4.0µL

incubate 16 °C 1 hour




Transformation

Tatsui

NameSampleCompetent CellsPlate
9/4 RBS-lysis1-DT 2µL20µLAmp
9/4 RBS-lysis2-DT 2µL20µLAmp
9/4 RBS-lysis3-DT 2µL20µLAmp 9/4 aptamer12-1R-DT2µL20µLAmp
9/4 pT181attenuator –pSB1C32µL20µLAmp
9/4 pT181antisense-pSB1C3 2µL20µLAmp
9/4 aptamer12-1R –pSB1C32µL20µLAmp
9/4 pSB4K5(2013plate5 56) 2µL20µLAmp
9/3 Mutagenesis product 1(KaiABC) 2µL20µLAmp
9/3 Mutagenesis product 2(NC) 2µL20µLAmp

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
DT193.11.931.81
Spinach-DT373.31.921.69
RBS-lysis1332.01.951.62

Master Plate

Hirano

NumberUse LB plate (+CP)
18/10 Plac(CP)-2
2 8/10 Plac(CP)-2
38/10 Plac(CP)-2

Master Plate

Hirano

NumberUse LB plate (+Amp)
18/9 J23100(Amp)-1
28/9 J23100(Amp)-1
38/9 J23100(Amp)-1


Liquid Culture

Hirano

Samplemedium
8/10 Plac(CP)-2Plusgrow medium(+CP)
8/9 J23100(Amp)-1Plusgrow medium(+Amp)

PCR

Hirano

genome DNAKOD plus10x bufferdNTPMgSO4F primer (RBS-luxI-DT-cloning-fond) R primer(RBS-luxI-DT-cloning-rex)MilliQtotal
8/29 pλ-RBS-luxI-DT0.3µL0.5µL 2.5µL 2.5µL 1.5µL 0.75µL 0.75µL 16.2µL 25µL
PreDenatureDenatureAnnealingExtensioncycle
 94°C 98°C 65°C 68°C--
5min10s 30s 3min10s 30cycles

PCR

Hirano

genome DNAKOD plus10x bufferdNTPMgSO4F primer (Pbad/araC-pSB1C3-cloning-fond) R primer(Pbad/araC-cloning-rex)MilliQtotal
8/31 Pbad/araC-RBS-RFP(1)0.3µL0.5µL 2.5µL 2.5µL 1.5µL 0.75µL 0.75µL 16.2µL 25µL
PreDenatureDenatureAnnealingExtensioncycle
 94°C 98°C 65°C 68°C--
5min10s 30s 3min10s 30cycles

Restriction Enzyme Digestion

No name

8/21 PconstSpeIPstIBufferBSAMilliQtotal
2 cuts10.5µL1.0µL1.0µL3.0µL0.3µL

14.2µL||30 µL

NC0.5&mic6/8ro;L0µL1.0µL0.1µL 8.4µL10 µL
8/29 PlacSpeIPstIBufferBSAMilliQtotal
2 cuts13.0µL1.0µL1.0µL3.0µL0.3µL

11.7µL||30 µL

NC0.7&mic6/8ro;L0µL1.0µL0.1µL 8.2µL10 µL
8/20 PtetSpeIPstIBufferBSAMilliQtotal
2 cuts9.0µL1.0µL1.0µL3.0µL0.3µL

15.7µL||30 µL

NC0.5&mic6/8ro;L0µL1.0µL0.1µL 8.4µL10 µL
8/20 RBS-tetR-DTXbaIPstIBufferBSAMilliQtotal
2 cuts8.5µL1.0µL1.0µL3.0µL0.3µL

16.2µL||30 µL

NC0.5&mic6/8ro;L0µL1.0µL0.1µL 8.4µL10 µL
spinach -DTXbaIPstIBufferBSAMilliQtotal
2 cuts5.4µL1.0µL1.0µL3.0µL3.0µL

16.6µL||30 µL

NC0.3&mic6/8ro;L0µL1.0µL1.0µL 7.7µL10 µL
8/17 RBS-lacZα-DT XbaIPstIBufferBSAMilliQtotal
2 cuts11µL1.0µL1.0µL3.0µL3.0µL

11µL||30 µL

NC0.5&mic6/8ro;L0µL1.0µL1.0µL 7.5µL10 µL

Transformation

No name

NameSampleCompetent CellsTotalPlate
Ptrc KaiC1µL10µL11µLAmp
Ptrc KaiB1µL10µL11µLAmp
pSω 1µL10µL11µLAmp

Electrophoresis

No name
1

LaneSampleEnzyme1Enzyme2
1100bp ladder---
2PconstSpeIPstI
3Pconst NC----
4PlacSpeIPstI
5Plac NC----
6100bp ladder----

File:Igku xxxxxx.xxx
2

LaneSampleEnzyme1Enzyme2
1100bp ladder---
2PtetSpeIPstI
3Ptet NC----
4RBS-tetR-DTXbaIPstI
5RBS-tetR-DT NC----
6100bp ladder----
File:Igku xxxxxx.xxx

Gel Extraction

No name

1

LaneDNAEnzyme
1100bp ladder--
2PconSpeI&PstI
3PconSpeI&PstI
4----
5PlacSpeI&PstI
6PlacSpeI&PstI

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Namequantityconcentration[µg/mL]260/280260/230
Pcon--13.2 1.99 -1.64
Plac-- 15.3 2.07 -o.39
No name

2

LaneDNAEnzyme
1100bp ladder--
2PtetSpeI&PstI
3PtetSpeI&PstI
4----
5RBS-tetR-DTXbaI&PstI
6RBS-tetR-DTXbaI&PstI

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Namequantityconcentration[µg/mL]260/280260/230
Ptet-- 30.1 1.97 -0.97
RBS-tetR-DT-- 3.0 2.44 0.88

Gel Extraction

No name


LaneDNAEnzyme
1100bp ladder--
2spinach-DTXbaII&PstI
3spinach-DT NCXbaII&PstI
4RBS-lacZα-DTXbaI&PstI
5RBS-lacZα-DT NCXbaI&PstI
6100bp ladder --

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Namequantityconcentration[µg/mL]260/280260/230
spinach-DT--2.1 2.10 -0.05
RBS-lacZα-DT-- 3.3 2.59 -0.07

Master Plate

No name

NumberUse LB plate (+Amp)
48/9 BBa_J23100-3
58/9 BBa_J23100-4
68/9 BBa_J23100-5

Master Plate

Hirano

NumberUse LB plate
1JM109-1
2JM109-2
3JM109-3
4JM109-4


Liquid Culture

Hirano

Samplemedium
8/9 BBa_J23100-3Plusgrow medium(+Amp)

Liquid Culture

Hirano

Samplemedium
JM109-1(Master plate)LB medium

Colony PCR

Hirano

Samplebase pair
8/9 BBa_J23100 3--
8/9 BBa_J23100 4--
8/9 BBa_J23100 5--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30s30cycles

Gel Extraction

No name


LaneDNAEnzyme
1100bp ladder--
2spinach-DTXbaII&PstI
3spinach-DT XbaII&PstI
4----
5RBS-lacZα-DTXbaI&PstI
6RBS-lacZα-DTXbaI&PstI

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Namequantityconcentration[µg/mL]260/280260/230
spinach-DT--2.1 2.10 -0.05
RBS-lacZα-DT-- 3.3 2.59 -0.07

</div>