Template:Kyoto/Notebook/Sep 13

From 2013.igem.org

(Difference between revisions)
(→Gel Extraction)
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<span class="author">No name</span>
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
-
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||SasA_fwd primer||SasA_rev primer||MilliQ||total
+
!genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total
|-
|-
-
| ||0.5||2.5||2.5||1.5||0.75||0.75|| ||25
+
|9/7 tetR  aptamer 12_1R(pSB1C3) ||0.5||6.05||1.75||1.7||0.5||0||10.5
 +
|}
 +
{| class="wikitable"
 +
!genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total
 +
|-
 +
|9/6 pT181 antisense-1(pSB1C3) ||0.5||6.05||1.75||1.7||0.5||0||10.5
 +
|}
 +
{| class="wikitable"
 +
!genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total
 +
|-
 +
|9/6 pT181 attenuator (pSB1C3)||0.5||3.85||1.75||3.9||0.5||0||10.5
 +
|}
 +
{| class="wikitable"
 +
!genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total
 +
|-
 +
|8/27 P&lambda;-RBS-luxI-DT-1||0.5||2.55||1.75||5.2||0.5||0||10.5
 +
|}
 +
{| class="wikitable"
 +
!genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total
 +
|-
 +
|8/31 Pbad/araC-RBS-RFP-2||0.5||3.75||1.75||4||0.5||0||10.5
 +
|}
 +
{| class="wikitable"
 +
!genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total
 +
|-
 +
|9/13 Plac(pSB1A2) ||0.5||4.2||1.75||3.5||0.5||0||10.5
 +
|}
 +
{| class="wikitable"
 +
!genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total
 +
|-
 +
|8/20 J23100-RBS-lacZ&alpha;-DT||0.5||6.35||1.75||1.4||0.5||10.5
 +
|}
 +
{| class="wikitable"
 +
!genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total
 +
|-
 +
|9/7 Ptet-RBS-lacZ&alpha;-DT||0.5||6.05||1.75||1.7||0.5||0||10.5
 +
|}
 +
{| class="wikitable"
 +
!genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total
 +
|-
 +
|9/8 J23100-spinach-DT||0.5||5.75||1.75||2||0.5||0||10.5
 +
|}
 +
{| class="wikitable"
 +
!genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total
 +
|-
 +
|9/11 RBS-lysis1-DT||0.5||6.05||1.75||1.7||0.5||10.5
 +
|}
 +
{| class="wikitable"
 +
!genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total
 +
|-
 +
|9/12 RBS-lysis2-DT||0.5||6.75||1.75||1.0||0.5||0||10.5
 +
|}
 +
{| class="wikitable"
 +
!genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total
 +
|-
 +
|9/12 RBS-lysis3-DT||0.5||6.75||1.75||1.0||0.5||0||10.5
 +
|}
 +
{| class="wikitable"
 +
!genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total
 +
|-
 +
|9/8 J23100-spinach-DT||0.5||6.35||1.75||1.4||0||0.5||10.5
 +
|}
 +
{| class="wikitable"
 +
!genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total
 +
|-
 +
|8/27 P&lambda;-RBS-luxI-DT-1||0.5||2.55||1.75||5.2||0||0.5||10.5
 +
|}
 +
{| class="wikitable"
 +
!genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total
 +
|-
 +
|9/12 RBS-lysis2-DT||0.5||6.75||1.75||1.0||0||0.5||10.5
 +
|}
 +
{| class="wikitable"
 +
!genome DNA||Big Dye||MilliQ||5x buffer||temp(400mg)||SasA_fwd primer||SasA_rev primer||total
 +
|-
 +
|9/11 RBS-lysis1-DT||0.5||6.05||1.75||1.7||0||0.5||10.5
|}
|}
{| class="wikitable"
{| class="wikitable"
!PreDenature||Denature||Annealing||Extension||cycle
!PreDenature||Denature||Annealing||Extension||cycle
|-
|-
-
|　&deg;C||　&deg;C||　&deg;C||　&deg;C||--
+
|96&deg;C||96&deg;C||50&deg;C||68&deg;C||--
|-
|-
-
|（◎Д◎）　||　||　||　||
+
|2m||10s||5s||4m||30
|}
|}
</div>
</div>

Revision as of 14:15, 25 September 2013

Contents

Sep 13

Miniprep

Nakamoto

DNAconcentration[µg/mL]260/280260/230
9/12 Fusion6 antisense245.91.881.41
9/12 Plux-3115.51.581.32
9/12 Plux-4125.31.931.44
9/12 Fusion3in2 attenuator-1231.91.811.31

Gel Extraction

Tatsui,Ashida

LaneDNAEnzyme
11kb ladder--
2RBS-lysis1-DTXbaI&PstI
3RBS-lysis1-DTXbaI&PstI
4RBS-lysis1-DTXbaI&PstI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(EcoRI&SpeI)9.81.500.39
Pcon-RBS-lacZα-DT(EcoRI&SpeI)11.21.470.08
LaneDNAEnzyme
11kb ladder--
3RBS-lysis2-DTXbaI&PstI
4RBS-lysis2-DTXbaI&PstI
5RBS-lysis2-DTXbaI&PstI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(EcoRI&SpeI)9.81.500.39
Pcon-RBS-lacZα-DT(EcoRI&SpeI)11.21.470.08

</div>

LaneDNAEnzyme
3RBS-lysis3-DTXbaI&PstI
4RBS-lysis3-DTXbaI&PstI
5RBS-lysis3-DTXbaI&PstI
61kb ladder--

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(EcoRI&SpeI)9.81.500.39
Pcon-RBS-lacZα-DT(EcoRI&SpeI)11.21.470.08

</div>

LaneDNAEnzyme
21kb ladder--
3pSB1C3EcoRI&SpeI
4pSB1C3EcoRI&SpeI
5pSB1C3EcoRI&SpeI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(EcoRI&SpeI)9.81.500.39
Pcon-RBS-lacZα-DT(EcoRI&SpeI)11.21.470.08

</div>

LaneDNAEnzyme
11kb ladder--
2pSB1C3XbaI&PstI
3pSB1C3XbaI&PstI
4pSB1C3XbaI&PstI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(EcoRI&SpeI)9.81.500.39
Pcon-RBS-lacZα-DT(EcoRI&SpeI)11.21.470.08

</div>

Restriction Enzyme Digestion

Nakamoto

9/13 PlacEcoRISpeIXbaIPstI BufferBSAMilliQtotal
1cuts(PstI)160µL0µL0µL1µL3µL3µL7µL30µL
NC0.80µL0µL0µL0µL1µL1µL7.2µL10µL
9/10 Pcon attenuatorEcoRISpeIXbaIPstIBufferBSAMilliQtotal
1cut(PstI)100µL0µL0µL1µL3µL3µL13µL30µL
NC0.50µL0µL0µL0µL1µL1µL7.5µL10µL
9/12 Plux EcoRISpeIXbaIPstI BufferBSAMilliQtotal
1cuts(PstI)120µL0µL0µL1µL3µL3µL11µL30µL
NC0.60µL0µL0µL0µL1µL1µL7.4µL10µL
9/12 DT-EEcoRISpeIXbaIPstIBufferBSAMilliQtotal
1cut(XbaI)23.70µL0µL1µL0µL3µL3µL0µL30.7µL
NC1.20µL0µL0µL0µL1µL1µL6.8µL10µL
9/12 Pcon-RBS-luxR-bTE EcoRISpeIXbaIPstI BufferBSAMilliQtotal
1cuts(XbaI)22.40µL0µL1µL0µL3µL3µL0.6µL30µL
NC2.20µL0µL0µL0µL1µL1µL5.8µL10µL
9/12 Pcon-PEcoRISpeIXbaIPstIBufferBSAMilliQtotal
1cut(SpeI)210µL1µL0µL0µL3µL3µL12.0µL30µL
NC2.10µL0µL0µL0µL1µL1µL5.7µL10µL

M9 (EDTA)

Kanbayashi

1mM EDTA
volume10ml
Na2HPO4 300mg
KH2PO4150mg
NaCl25mg
NH4Cl50mg
Fe(3)-EDTA21mg　

5mM EDTA</div>

volume10ml
Na2HPO4300mg
KH2PO4150mg
NaCl25mg
NH4Cl50mg
Fe(3)-EDTA105mg　

</div>

Gel Extraction

Nakamoto

LaneDNAEnzyme
11kb ladder--
3Pcon-PSpeI
4Pcon-PSpeI
5Pcon-PSpeI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(EcoRI&SpeI)9.81.500.39
Pcon-RBS-lacZα-DT(EcoRI&SpeI)11.21.470.08

Liquid Culture

Tatsui

Samplemedium
9/11 aptamer12_1M(pSB1C3)-1--
9/11 aptamer 12_P1--
9/11 Fusaion1 antisense-4--
9/12 pSB4K5-1--
9/12 pSB4K5-2--

Restriction Enzyme Digestion

Nakamoto

9/13 Plac-PEcoRISpeIXbaIPstI BufferBSAMilliQtotal
1cuts(SpeI)23.50µL0.5µL0µL0µL3µL3µL0µL30µL
NC80µL0µL0µL0µL1µL1µL0µL10µL
9/13 Pcon-pT181 attenuator-PEcoRISpeIXbaIPstIBufferBSAMilliQtotal
1cut(SpeI)23.50µL0.5µL0µL0µL3µL3µL0µL30µL
NC02.30µL0µL0µL0µL1µL1µL5.7µL10µL
9/13 Plux-P EcoRISpeIXbaIPstI BufferBSAMilliQtotal
1cuts(SpeI)23.80µL0.2µL0µL1µL3µL3µL0µL30µL
NC80µL0µL0µL0µL1µL1µL0µL10µL
9/5 RBS-lacZα-DTEcoRISpeIXbaIPstIBufferBSAMilliQtotal
2cut(XbaI&PstI)10.80µL0µL1µL1µL3µL3µL11.2µL30µL
NC0.50µL0µL0µL0µL1µL1µL7.5µL10µL

Electrophoresis

Hirano

LaneSample
1100bp ladder
2RBS-lacZα-DT(XbaI&PstI)
3RBS-lacZα-DT(NC9

File:Igku xxxxxxx.xxx

Ligation

No Name

stateVectorInserterLigation High ver.2
experiment9/13 Plac (SpeI&PstI)8.5µL9/13 pT181 antisense (XbaI&PstI)2.1 µL10.6 µL
experiment9/13 Plac (SpeI&PstI)8.5 µL9/10 aptamer 12_1R-DT (XbaI&PstI)1.8 µL10 .3µL
experiment9/13 Plac (SpeI&PstI)8.5 µL9/2 pT181 attenuator(XbaI&PstI)2.3 µL10.8 µL
experiment9/13 Plux (SpeI&PstI)11.4 µL9/13 RBS-lysis1-DT (XbaI&PstI)5.0 µL16.4 µL
experiment9/13 Plux (SpeI&PstI)11.4 µL9/13 RBS-lysis2-DT (XbaI&PstI)7.0 µL18.4 µL
experiment9/13 Plux (SpeI&PstI)7.2 µL9/13 RBS-lysis3-DT (XbaI&PstI)6.0 µL13.2 µL
experiment9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI)2.0 µL9/4 Plux-RBS-GFP-DT (EcoRI&SpeI)5.2 µL72 µL
experiment9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI)2.0 µL9/10 aptamer 12_1R-DT (XbaI&PstI)1.6 µL3.6 µL
experiment9/12 Ptet-pT181 antisense (--)1.0 µL9/10 spinach-DT (--)2.4 µL3.4 µL

PCR

No name

genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/7 tetR aptamer 12_1R(pSB1C3) 0.56.051.751.70.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/6 pT181 antisense-1(pSB1C3) 0.56.051.751.70.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/6 pT181 attenuator (pSB1C3)0.53.851.753.90.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
8/27 Pλ-RBS-luxI-DT-10.52.551.755.20.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
8/31 Pbad/araC-RBS-RFP-20.53.751.7540.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/13 Plac(pSB1A2) 0.54.21.753.50.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
8/20 J23100-RBS-lacZα-DT0.56.351.751.40.510.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/7 Ptet-RBS-lacZα-DT0.56.051.751.70.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/8 J23100-spinach-DT0.55.751.7520.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/11 RBS-lysis1-DT0.56.051.751.70.510.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/12 RBS-lysis2-DT0.56.751.751.00.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/12 RBS-lysis3-DT0.56.751.751.00.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/8 J23100-spinach-DT0.56.351.751.400.510.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
8/27 Pλ-RBS-luxI-DT-10.52.551.755.200.510.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/12 RBS-lysis2-DT0.56.751.751.000.510.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/11 RBS-lysis1-DT0.56.051.751.700.510.5
PreDenatureDenatureAnnealingExtensioncycle
96°C96°C50°C68°C--
2m10s5s4m30

PCR

No name

genome DNAKOD plus10x bufferdNTPMgSO4SasA_fwd primerSasA_rev primerMilliQtotal
0.52.52.51.50.750.75 25
PreDenatureDenatureAnnealingExtensioncycle
　°C　°C　°C　°C--
（◎Д◎）　　　　

Gel Extraction

Nakamoto

LaneDNAEnzyme
1100bp ladder--
3RBS-lacZα-DTXbaI&PstI
4RBS-lacZα-DTXbaI&PstI
5RBS-lacZα-DTXbaI&PstI
6RBS-lacZα-DTXbaI&PstI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(XbaI&PstI)9.81.500.39

Transformation

No name

NameSampleCompetent CellsTotalPlate
9/13 Plac+pT181 antisense2µL20µL22µLAmp
9/13 Plac+apt12_1R2µL20µL22µLAmp
9/13 Plac+pT181 attenuator2µL20µL22µLAmp
9/13 Plux+pT181 antisense2µL20µL22µLCP
9/13 Plux+RBS-lysis2-DT2µL20µL22µLCP
9/13 Plux+RBS-lysis2-DT2µL20µL22µLCP
9/13 Pcon-RBS-lacR-DT+Plux-RBS-GFP-DT2µL20µL22µLAmp
9/13 Pcon-pT181 antisense+apt12_1R-DT2µL20µL22µLAmp
9/13 Ptet-pT181antisense+spinach-DT2µL20µL22µLCP
Pλ-luxI-12µL20µL22µLAmp
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