Template:Kyoto/Notebook/Sep 14

From 2013.igem.org

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(→Transformation)
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===PCR===
===PCR===
<div class="experiment">
<div class="experiment">
-
<span class="author">No name</span>
+
<span class="author">Tatsui</span>
{| class="wikitable"
{| class="wikitable"
-
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||SasA_fwd primer||SasA_rev primer||MilliQ||total
+
!genome DNA||temp||Big Dye||5x buffer||primer||MilliQ||total
|-
|-
-
| ||0.5||2.5||2.5||1.5||0.75||0.75|| ||25
+
|9/6 PT181 attenuator(VR)||3.9||0.5||1.75||0.5||3.85||10.5
 +
|-
 +
|8/17 RBS lacZα-DT||2.1||0.5||1.75||0.5||3.85||10.5
 +
|-
 +
|9/6 PT181 attenuator(VR)||3.9||0.5||1.75||0.5||3.85||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||50&deg;C||60&deg;C||4&deg;C||--
|-
|-
-
|（◎Д◎）　||　||　||　||
+
|2min||13s||5s||4min||30cycle
|}
|}
</div>
</div>

Revision as of 14:56, 25 September 2013

Contents

Sep 14

Restriction Enzyme Digestion

Hirano

9/14 pSB4K5SEcoRIXbaISpeIPstIBufferBBufferDBSAMilliQtotal
2cuts10µL1µL0µL1µL0µL3µL0µL3µL12µL30µL
NC0.5µL0µL0µL0µL0µL1µL0µL1µL7.5µL10µL
9/13 PlacXbaIPstIBufferHBSAMilliQtotal
1 cut9µL0µL1µL3µL3µL14µL30µL
NC1µL0µL0µL1µL1µL7µL10µL
9/14 PKaiBCEcoRIXbaISpeIPstIBufferBBSAMilliQtotal
2cuts11µL1µL0µL1µL0µL3µL3µL11µL30µL
NC4µL0µL0µL0µL0µL1µL1µL4µL10µL
9/14 RpaBEcoRIXbaISpeIPstIBufferDBSAMilliQtotal
2cuts13µL0µL1µL0µL1µL3µL3µL9µL30µL
NC2µL0µL0µL0µL0µL1µL1µL5µL10µL
8/17 RBS-GFP-DTEcoRIXbaISpeIPstIBufferDBSAMilliQtotal
1cut9µL0µL1µL0µL0µL3µL3µL14µL30µL
NC0.5µL0µL0µL0µL0µL1µL1µL7.5µL10µL
  • incubated 37°C 1hour

Liquid Culture

Hirano

Samplemedium
9/11 Plac(BBa-R0011)-(3)Plusgrow medium(+Amp)

PCR

No name

genome DNAMilliQBig Dye5x buffertemp(400mg)primertotal
Pcon-RBS-luxR-DT0.51.751.750.40.55.110.5
Pcon-RBS-luxR-DT0.51.751.750.40.55.110.5
Pcon-RBS-tetR-DT0.51.751.7530.5310.5
Pcon-RBS-tetR-DT0.51.751.7530.5310.5
Pcon-RBS-luxR-DT0.51.751.752.40.53.610.5
Plux-RBS-GFP-DT0.51.751.751.90.54.110.5
Plux-RBS-GFP-DT0.51.750.51.90.54.110.5
Pcon-RBS-GFP-DT0.51.750.51.20.54.810.5
RBS-lysis3-DT0.51.750.51.00.55.010.5
Pcon-RBS-GFP-DT0.51.750.51.40.54.610.5
Pcon-RBS-GFP-DT0.51.750.51.40.54.610.5
RBS-lysis3-DT0.51.751.750.40.55.110.5
Ptet-RBS-spinach-DT0.51.750.50.590.50.110.5
PreDenatureDenatureAnnealingExtensioncycle
96°C96°C50°C60°C--
2min10s5s4min30cycle

Restriction Enzyme Digestion

No name

9/14 Plac(1A2)EcoRIXbaISpeIPstIBufferBBSAMilliQtotal
2cuts23µL0µL0µL0.3µL0µL3µL3µL0µL30µL
9/14 RBS-GFP-DTEcoRIXbaISpeIPstIBufferHBSAMilliQtotal
2 cuts23µL1µL0µL0µL0µL3µL3µL0µL30µL

Ligation

Nakamoto and Hirano

stateVectorInserterLigation High ver.2
experiment9/13 Pcon-PT181attenuater(SpeI&PstI)1.79/13 RBS-lacZα-DT (XbaI & PstI)12.55
experiment9/13 pSB2C3 (XbaI & PstI)1.99/14 RpaB (XbaI & PstI)16.320.1
experiment9/13 pSB2C3 (XbaI & PstI)4.79/14 PkaiBC (EcoRI & SpeI)6.711.4
experiment9/14 RBS-GFP-DT (PstI&XbaI)0.79/14 PkaiBC (EcoRI & SpeI)2.63.3
experiment9/14 Plac (SpeI&PstI)2.49/14 RBS-lysis1-DT (XbaI & PstI)4.97.3
experiment9/14 Plac (SpeI&PstI)2.49/14 RBS-lysis2-DT (XbaI & PstI)7.09.4
experiment9/14 Plac (SpeI&PstI)2.49/14 RBS-lysis3-DT (XbaI & PstI)9.211.6

Samples were evaporeted used evaporator into about 7 µL

Colony PCR

No name

Samplebase pair
9/13 Plux＋RBS-lysis1-DT (1)~(3)--
9/13 Plux＋RBS-lysis2-DT (1)~(3)-
9/13 Plux＋RBS-lysis3-DT (1)~(2)-
Samplebase pair
9/13 Pcon-RBS-luxR-DT＋Plux-RBS-GFP-DT (1)~(4)--
9/13 Pcon-PT181attenuator＋aptamer121R-DT (1)~(2)-
9/13 Plac＋PT181attenuator (1)-
Samplebase pair
Dλ-lux(1) (1)~(2)--
NC --

Liquid Culture

No name

Samplemedium
9/13 Pλ-luxI(1)-1Plusgrow medium(+Amp)

Transformation

Nakamoto

NameSampleCompetent CellsTotalPlate
9/13 RBS-lysisZα-DT(XbaI&PstI)＋9/13 Pcon-PT181 attenuator(SpeI&PstI)2µL20µL22µLAmp
9/13 SB2C3＋9/14 PzαB2µL20µL22µLCP
9/13 pSB2C3＋9/14 PkaiBC2µL20µL22µLCP
9/13 RBS-GFP-DT＋9/14 PkaiBC2µL20µL22µLCP
Plac＋RBS-lysis1-DT2µL20µL22µLAmp
Plac＋RBS-lysis2-DT2µL20µL22µLAmp
Plac＋RBS-lysis2-DT2µL20µL22µLAmp

PCR

Tatsui

genome DNAtempBig Dye5x bufferprimerMilliQtotal
9/6 PT181 attenuator(VR)3.90.51.750.53.8510.5
8/17 RBS lacZα-DT2.10.51.750.53.8510.5
9/6 PT181 attenuator(VR)3.90.51.750.53.8510.5
PreDenatureDenatureAnnealingExtensioncycle
96°C50°C60°C4°C--
2min13s5s4min30cycle
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