Template:Kyoto/Notebook/Sep 6

From 2013.igem.org

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!|Sample||base pair
!|Sample||base pair
|-
|-
-
|8/27 Pcon-pT181 antisense-(1)||542
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|Pconst-spinach-DT-(1~4)||563
|-
|-
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|8/26 pT181 attenuator-DT-(1)||738
+
|Plac-spinach-DT-(1~4)||659
 +
|-
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|Pcon-PT181 antisense-(1~4)||468
 +
|-
 +
|Ptet-PT181 antisense-(1~4)||604
 +
|-
 +
|Pconst-PT181 attenuater-(1~4)||568
 +
|-
 +
|Pconst＋RBS-tetR-DT-(1~4)||1121
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|-
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|Ptet＋RBS-lacZα-DT-(1~4)||1162
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{| class="wikitable"
{| class="wikitable"
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|94°C||94°C||55°C||68°C||--
|94°C||94°C||55°C||68°C||--
|-
|-
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|5min||30s||30s||30s||30cycles
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|5min||30s||30s||40s||30cycles
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[[File:igku_Aug19electrophoresis1.png]]
 
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Revision as of 11:30, 24 September 2013

Contents

Sep 6

Liquid Culture

No name

Samplemedium
9/4 RBS-Lysis3＋DT3Plusgrow medium(+Cp)
9/4 RBS-Lysis1＋DT3Plusgrow medium(+Cp)
9/4 PSB4K5Plusgrow medium(+K)　

Colony PCR

No name

Samplebase pair
Pconst-spinach-DT-(1)-
Pconst-spinach-DT-(2)-
Pconst-spinach-DT-(3)-
Pconst-spinach-DT-(4)-
Plac-spinach-DT-(1)-
Plac-spinach-DT-(2)-
Plac-spinach-DT-(3)-
Plac-spinach-DT-(4)-
Pcon-PT181 antisense-(1)-
Pcon-PT181 antisense-(2)-
Pcon-PT181 antisense-(3)-
Pcon-PT181 antisense-(4)-
Samplebase pair
Ptet-PT181 antisense-(1)-
Ptet-PT181 antisense-(2)-
Ptet-PT181 antisense-(3)-
Ptet-PT181 antisense-(4)-
Plac-PT181 attenuater-(1)-
Plac-PT181 attenuater-(2)-
Plac-PT181 attenuater-(3)-
Plac-PT181 attenuater-(4)-

Miniprep

Honda and Kojima

DNAconcentration [µg/mL]260/280260/230
PT181 antisense-(1)238.71.681.44
PT181 antisense-(2)248.81.831.77
PT181 antisense-(3)223.21.781.61
PT181 antisense-(4)220.81.891.92
tetR aptamer-(1)252.51.891.90
tetR aptamer-(2)211.21.901.99
DT181 attenuater102.21.982.17
121R aptamer-DT251.71.922.01
PSB4K5212.91.801.90
RBS-lacZα-DT185.71.781.79
RBS-lysis2-DT-(1)84.81.852.24

Restriction Enzyme Digestion

Tatsui

9/6 RBS-lysis2-DT (78.3 µg/mL)EcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts22µL0µL0µL1µL1µL3µL3µL0µL30µL
NC1.3µL0µL0µL0µL0µL1µL1µL6.7µL10µL
  • at 37°C, for 1h
aptamer-DT (251.7 µg/mL)EcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts0.4µL0µL0µL1µL1µL3µL3µL14.1µL30µL
NC0.4µL0µL0µL0µL0µL1µL1µL7.6µL10µL
  • at 37°C, for 1h
Pλ-luxI (86.4 µg/mL)EcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts23.1µL1µL1µL0µL0µL3µL3µL1.9µL30µL
NC1.2µL0µL0µL0µL0µL1µL1µL6.8µL10µL
  • at 37°C, for 1h

Colony PCR

Hirano

Samplebase pair
Pconst＋RBS-tetR-DT-(1)1121
Pconst＋RBS-tetR-DT-(2)1121
Pconst＋RBS-tetR-DT-(3)1121
Pconst＋RBS-tetR-DT-(4)1121
Ptet＋RBS-lacZα-DT-(1)1162
Ptet＋RBS-lacZα-DT-(2)1162
Ptet＋RBS-lacZα-DT-(3)1162
Ptet＋RBS-lacZα-DT-(4)1162

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
2RBS-lysis2-DTXbaI & PstI
3
4----
5aptamer 12-1R-DTXbaI & PstI
6
LaneDNAEnzyme
1100bp ladder--
2Pλ-lux3EcoRI & SpeI
3
4
5
6
Nameconcentration[µg/mL]260/280260/230
RBS-lysis2-DT (XbaI&PstI)7.11.880.07
aptamer 12-1R-DT (XbaI&PstI)15.31.930.70
Pλ-lux3 (EcoRI&SpeI)4.51.790.31

Liquid Culture

No name

Samplemedium
9/5　Pconst-spinach-DT-(1)Plusgrow medium(+Amp&CP)
9/5　Ptet-PT181 antisense-(1)Plusgrow medium(+CP)
9/5　Pconst＋RBS-tetR-DT-(1)Plusgrow medium(+Amp&CP)
9/5　Ptet＋RBS-lacZα-DT-(1)Plusgrow medium(+CP)
9/5　Pcon-PT181 attenuater-(1)Plusgrow medium(+CP)
9/4　pT181 antisense-(1)Plusgrow medium(+CP)
9/4　aptamer 12-1R-(1)Plusgrow medium(+CP)

Colony PCR

No name

Samplebase pair
9/5 Plac-spinach-DT-(5~12)563
9/5 Pcon-antisense-(5~12)468
9/5 Plac-attenuater-(5~12)664
9/5 PT181 antisense＋Plac-(1)-
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s42s30cycles

Colony PCR

No name

Samplebase pair
Pconst-spinach-DT-(1~4)563
Plac-spinach-DT-(1~4)659
Pcon-PT181 antisense-(1~4)468
Ptet-PT181 antisense-(1~4)604
Pconst-PT181 attenuater-(1~4)568
Pconst＋RBS-tetR-DT-(1~4)1121
Ptet＋RBS-lacZα-DT-(1~4)1162
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s40s30cycles
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