Template:Kyoto/Notebook/Sep 14

From 2013.igem.org

(Difference between revisions)
(→Restriction Enzyme Digestion)
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!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
|-
|-
-
|experiment||8/30 pSB1C3 (EcoRI & SpeI)||6.6||8/30 tRNA-Spinach (EcoRI & SpeI)||12.5||3.5
+
|experiment||9/13 Pcon-PT181attenuater(SpeI&PstI)||1.7||9/13 RBS-lacZα-DT (XbaI & PstI)||12.5||5
|-
|-
-
|experiment||8/30 pSB1C3 (EcoRI & SpeI)||6.6||8/30 pT181 antisense (EcoRI & SpeI)||12.5||3.5
+
|experiment||9/13 pSB2C3 (XbaI & PstI)||1.9||9/14 RpaB (XbaI & PstI)||16.3||20.1
|-
|-
-
|experiment||8/30 pSB1C3 (XbaI & Pst)||12.5||8/30 pT181 attenuator (XbaI & PstI)||25||3.5
+
|experiment||9/13 pSB2C3 (XbaI & PstI)||4.7||9/14 PkaiBC (EcoRI & SpeI)||6.7||11.4
|-
|-
-
|experiment||8/30 pSB1C3 (XbaI & Pst)||12.5||8/30 pT181 antisense (XbaI & PstI)||25||3.5
+
|experiment||9/14 RBS-GFP-DT (PstI&XbaI)||0.7||9/14 PkaiBC (EcoRI & SpeI)||2.6||3.3
|-
|-
-
|experiment||8/20 DT (EcoRI & XbaI)||6.6||8/30 pT181 attenuator(2) (EcoRI & SpeI)||12.5||3.5
+
|experiment||9/14 Plac (SpeI&PstI)||2.4||9/14 RBS-lysis1-DT (XbaI & PstI)||4.9||7.3
|-
|-
-
|experiment||8/20 DT (EcoRI & XbaI)||6.6||8/30 pT181 antisense (EcoRI & SpeI)||12.5||3.5
+
|experiment||9/14 Plac (SpeI&PstI)||2.4||9/14 RBS-lysis2-DT (XbaI & PstI)||7.0||9.4
|-
|-
-
|experiment||8/27 DT (EcoRI & XbaI)||3.0||8/30 tRNA-Spinach (EcoRI & SpeI)||12.5||3.5
+
|experiment||9/14 Plac (SpeI&PstI)||2.4||9/14 RBS-lysis3-DT (XbaI & PstI)||9.2||11.6
-
|-
+
-
|experiment||8/20 DT (EcoRI & XbaI)||6.6||8/30 RBS-lysis2 (EcoRI & SpeI)||12.5||3.5
+
-
|-
+
-
|experiment||8/22 J23100 (SpeI & PstI)||--||RBS-tetR-DT (XbaI & PstI)||--||3.5
+
-
|-
+
-
|experiment||8/22 Plac (SpeI & PstI)||8.0||8/18 RBS-lacZα-DT (XbaI & PstI)||10||3.5
+
|}
|}
Samples were evaporeted used evaporator into about 7 µL
Samples were evaporeted used evaporator into about 7 µL
</div>
</div>
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===Colony PCR===
===Colony PCR===

Revision as of 13:56, 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
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT(8/20 LB Amp) (1)~(4)2143
8/29 Plux-RBS-GFP-DT(8/27 LB-CP) (1)~(2)1227
8/29 Pbad/araC-RBS-RFP{BBa_I13516} (8/21 LB-CP) (1)~(2)2256
8/29 RBS-lysis3-DT-(1)1210
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s2min30cycle

Liquid Culture

No name

Samplemedium
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1)Plusgrow medium(+Amp)
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(2)Plusgrow medium(+Amp)
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(3)Plusgrow medium(+Amp)
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(4)Plusgrow medium(+Amp)
8/29 Plux-RBS-GFP-DT-(1)Plusgrow medium(+CP)
8/29 Plux-RBS-GFP-DT-(2)Plusgrow medium(+CP)
8/29 Pbad/araC-RBS-RFP-DT-(1)Plusgrow medium(+CP)
8/29 Pbad-araC-RBS-RFP-DT-(2)Plusgrow medium(+CP)
RBS-lysis3-DT-(1)Plusgrow medium(+CP)

Transformation

No Name

NameSampleCompetent CellsTotalPlate
8/27 pT181 attenuator-DT2µL20µL22µLCP
8/27 RBS-lysis3-DT2µL20µL22µLCP
8/28 RBS-lysis3-DT2µL20µL22µLCP
8/28 Pcon-RBS-GFP-DT-Pcon-RBS-LuxR-DT2µL20µL22µLAmp
8/28 RBS-lysis1-DT2µL20µL22µLCP
8/28 Plux-RBS-GFP-DT2µL20µL22µLCP
8/28 Spinach-DT2µL20µL22µLCP

Igku Aug29 Transformation1(N④-1).jpg
Igku Aug29 Transformation(N④-2).jpg
Igku Aug29 Transformation(N⑤).jpg
File:Igku Aug29 Transformation()after


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◎）　　　　
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