Template:Kyoto/Notebook/Sep 14

From 2013.igem.org

(Difference between revisions)
(→PCR)
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<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
-
! ||8/22 psB1C3-(2)||EcoRI||SpeI||XbaI||PstI||BufferB||BufferD||BSA||MilliQ||total
+
! ||9/14 Plac(1A2)||EcoRI||XbaI||SpeI||PstI||BufferB||BSA||MilliQ||total
|-
|-
-
|2 cuts||8&micro;L||0.5&micro;L||0.5&micro;L||0&micro;L||0&micro;L||3&micro;L||0&micro;L||0.3&micro;L||17.7&micro;L||30&micro;L
+
|2cuts||23&micro;L||0&micro;L||0&micro;L||0.3&micro;L||0&micro;L||3&micro;L||3&micro;L||0&micro;L||30&micro;L
-
|-
+
-
|NC||1&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0&micro;L||0.1&micro;L||7.9&micro;L||10&micro;L
+
-
|-
+
-
|2 cuts||8&micro;L||0&micro;L||0&micro;L||0.5&micro;L||0.5&micro;L||0&micro;L||3&micro;L||0.3&micro;L||17.7&micro;L||30&micro;L
+
-
|-
+
-
|NC||1&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||7.9&micro;L||10&micro;L
+
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||8/21 tRMA-spinach(1)||XbaI||SpeI||BufferB||BSA||MilliQ||total
+
! ||9/14 RBS-GFP-DT||EcoRI||XbaI||SpeI||PstI||BufferH||BSA||MilliQ||total
-
|-
+
-
|2 cuts||4&micro;L||0.5&micro;L||0.5&micro;L||3&micro;L||0.3&micro;L||21.7&micro;L||30&micro;L
+
-
|-
+
-
|NC||0.5&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.4&micro;L||10&micro;L
+
-
|}
+
-
{| class="wikitable"
+
-
! ||8/21 tetR aptamer12-1R(1)||EcoRI||SpeI||BufferB||BSA||MilliQ||total
+
-
|-
+
-
|2 cuts||3&micro;L||0.5&micro;L||0.5&micro;L||3&micro;L||0.3&micro;L||22.7&micro;L||30&micro;L
+
-
|-
+
-
|NC||0.4&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.5&micro;L||10&micro;L
+
-
|}
+
-
{| class="wikitable"
+
-
! ||8/21 PT181 attenuator-(2)||EcoRI||SpeI||XbaI||PstI||BufferB||BufferD||BSA||MilliQ||total
+
-
|-
+
-
|2 cuts||4&micro;L||0.5&micro;L||0.5&micro;L||0&micro;L||0&micro;L||3&micro;L||0&micro;L||0.3&micro;L||21.7&micro;L||30&micro;L
+
-
|-
+
-
|NC||0.5&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0&micro;L||0.1&micro;L||8.4&micro;L||10&micro;L
+
-
|-
+
-
|2 cuts||4&micro;L||0&micro;L||0&micro;L||0.5&micro;L||0.5&micro;L||0&micro;L||3&micro;L||0.3&micro;L||21.7&micro;L||30&micro;L
+
-
|-
+
-
|NC||0.5&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.4&micro;L||10&micro;L
+
-
|}
+
-
{| class="wikitable"
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! ||8/21 PT181 antisense-(2)||EcoRI||SpeI||XbaI||PstI||BufferB||BufferD||BSA||MilliQ||total
+
-
|-
+
-
|2 cuts||10&micro;L||0.5&micro;L||0.5&micro;L||0&micro;L||0&micro;L||3&micro;L||0&micro;L||0.3&micro;L||15.7&micro;L||30&micro;L
+
-
|-
+
-
|NC||1&micro;L||0&micro;L||0&;L||0&micro;L||0&micro;L||1&micro;L||0&micro;L||0.1&micro;L||7.9&micro;L||10&micro;L
+
-
|-
+
-
|2 cuts||10&micro;L||0&micro;L||0&micro;L||0.5&micro;L||0.5&micro;L||0&micro;L||3&micro;L||0.3&micro;L||15.7&micro;L||30&micro;L
+
|-
|-
-
|NC||1&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||7.9&micro;L||10&micro;L
+
|2 cuts||23&micro;L||1&micro;L||0&micro;L||0&micro;L||0&micro;L||3&micro;L||3&micro;L||0&micro;L||30&micro;L
|}
|}
-
*incubated 37&deg;C 1hour
 
-
[[File:Igku Aug29 Restriction Enzyme Digestion(N③).jpg]]<br>
 
</div>
</div>
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===Ligation===  
===Ligation===  

Revision as of 11:14, 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
experiment8/30 pSB1C3 (EcoRI & SpeI)6.68/30 tRNA-Spinach (EcoRI & SpeI)12.53.5
experiment8/30 pSB1C3 (EcoRI & SpeI)6.68/30 pT181 antisense (EcoRI & SpeI)12.53.5
experiment8/30 pSB1C3 (XbaI & Pst)12.58/30 pT181 attenuator (XbaI & PstI)253.5
experiment8/30 pSB1C3 (XbaI & Pst)12.58/30 pT181 antisense (XbaI & PstI)253.5
experiment8/20 DT (EcoRI & XbaI)6.68/30 pT181 attenuator(2) (EcoRI & SpeI)12.53.5
experiment8/20 DT (EcoRI & XbaI)6.68/30 pT181 antisense (EcoRI & SpeI)12.53.5
experiment8/27 DT (EcoRI & XbaI)3.08/30 tRNA-Spinach (EcoRI & SpeI)12.53.5
experiment8/20 DT (EcoRI & XbaI)6.68/30 RBS-lysis2 (EcoRI & SpeI)12.53.5
experiment8/22 J23100 (SpeI & PstI)--RBS-tetR-DT (XbaI & PstI)--3.5
experiment8/22 Plac (SpeI & PstI)8.08/18 RBS-lacZα-DT (XbaI & PstI)103.5

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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