Template:Kyoto/Notebook/Sep 6

From 2013.igem.org

(Difference between revisions)
(→Restriction Enzyme Digestion)
(→Colony PCR)
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*at 37 °C, for 1h
*at 37 °C, for 1h
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===Colony PCR===
 
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<div class="experiment">
 
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<span class="author">Hirano</span>
 
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{| class="wikitable"
 
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!|Sample||base pair
 
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|-
 
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|Pconst+RBS-tetR-DT-(1)||1121
 
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|-
 
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|Pconst+RBS-tetR-DT-(2)||1121
 
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|-
 
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|Pconst+RBS-tetR-DT-(3)||1121
 
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|-
 
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|Pconst+RBS-tetR-DT-(4)||1121
 
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|-
 
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|Ptet+RBS-lacZ&alpha;-DT-(1)||1162
 
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|-
 
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|Ptet+RBS-lacZ&alpha;-DT-(2)||1162
 
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|-
 
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|Ptet+RBS-lacZ&alpha;-DT-(3)||1162
 
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|-
 
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|Ptet+RBS-lacZ&alpha;-DT-(4)||1162
 
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|}
 
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</div>
 
===Gel Extraction===
===Gel Extraction===

Revision as of 15:49, 24 September 2013

Contents

Sep 6

Electrophoresis

Hirano

LaneSample
1100bp ladder
29/4 RBS-lysis3-DT -3
39/4 RBS-lysis3-DT -4
49/4 RBS-lysis3-DT -5
59/4 RBS-lysis1-DT -3
69/4 Ptrc-KaiC -1
79/4 pSB4K5 -1
8100bp ladder

File:Igku xxxxxx.xxx

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


Liquid Culture

No name

Samplemedium
9/4 RBS-Lysis3+DT3Plusgrow medium(+CP)
9/4 RBS-Lysis1+DT3Plusgrow medium(+CP)
9/4 pSB4K5Plusgrow medium(+Kan)

Electrophoresis

No name

LaneSample
1100bp ladder
2Pconst-spinach-DT -(1)
3Pconst-spinach-DT -(2)
4Pconst-spinach-DT -(3)
5Pconst-spinach-DT -(4)
6Plac-spinach-DT -(1)
7Plac-spinach-DT -(2)
8Plac-spinach-DT -(3)
9Plac-spinach-DT -(4)
10Pcon-pT181 antisense -(1)
11Pcon-pT181 antisense -(2)
12Pcon-pT181 antisense -(3)
13Pcon-pT181 antisense -(4)

File:Igku xxxxxx.xxx

LaneSample
1100bp ladder
2Ptet-pT181 antisense -(1)
3Ptet-pT181 antisense -(2)
4Ptet-pT181 antisense -(3)
5Ptet-pT181 antisense -(4)
6Plac-pT181 attenuater -(1)
7Plac-pT181 attenuater -(2)
8Plac-pT181 attenuater -(3)
9Plac-pT181 attenuater -(4)

File:Igku xxxxxx.xxx

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)XbaIPstIBSABufferMilliQtotal
2 cuts22 µL1 µL1 µL3 µL3 µL0 µL30 µL
NC1.3 µL0 µL0 µL1 µL1 µL6.7 µL10 µL
aptamer-DT (251.7 µg/mL)XbaIPstIBSABufferMilliQtotal
2 cuts0.4 µL1 µL1 µL3 µL3 µL14.1 µL30 µL
NC0.4 µL0 µL0 µL1 µL1 µL7.6 µL10 µL
Pλ-luxI (86.4 µg/mL)EcoRISpeIBSABufferMilliQtotal
2 cuts23.1 µL1 µL1 µL3 µL3 µL1.9 µL30 µL
NC1.2 µL0 µL0 µL1 µL1 µL6.8 µL10 µL
  • at 37 °C, for 1h

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
2RBS-lysis2-DTXbaI & PstI
3
4----
5aptamer 12_1R-DTXbaI & PstI
6
LaneDNAEnzyme
1100bp ladder--
2Pλ-luxIEcoRI & SpeI
3
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-luxI (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
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