Template:Kyoto/Notebook/Sep 11

From 2013.igem.org

(Difference between revisions)
(→Restriction Enzyme Digestion)
(→Restriction Enzyme Digestion)
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<span class="author">Nakamoto</span>
<span class="author">Nakamoto</span>
{| class="wikitable"
{| class="wikitable"
-
! ||DNA||E||X||S||P||buffer||BSA||MilliQ||Total||
+
! ||DNA||EcoRl||Xbal||Stel||Pstl||buffer||BSA||MilliQ||Total||
|-
|-
-
|pSB4K5(EcoR1+S)||9.4&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 3&micro;L|| 3&micro;L|| 12.6&micro;L||30 &micro;L
+
|pSB4K5(EcoRl+Stel)||9.4&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 3&micro;L|| 3&micro;L|| 12.6&micro;L||30 &micro;L
|-
|-
-
|pSB4K5(E+S) negative||0.5&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 7.5&micro;L|| 10&micro;L
+
|pSB4K5(EcoRl+Stel) negative||0.5&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 7.5&micro;L|| 10&micro;L
|-
|-
-
|RBS-lacZ&alpha;-DT(E+S)|| 10&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 3&micro;L|| 3&micro;L|| 12&micro;L|| 30&micro;L
+
|RBS-lacZ&alpha;-DT(EcoRl+Stel)|| 10&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 3&micro;L|| 3&micro;L|| 12&micro;L|| 30&micro;L
|-
|-
-
|RBS-lacZ&alpha;-DT(E+S) negative|| 0.5&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 7.5&micro;L|| 10&micro;L
+
|RBS-lacZ&alpha;-DT(EcoRl+Stel) negative|| 0.5&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 7.5&micro;L|| 10&micro;L
|-
|-
-
|Ptet-pT181 antisense(S+P)|| 10.8&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L|| 12&micro;L|| 30&micro;L
+
|Ptet-pT181 antisense(Stel+Pstl)|| 10.8&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L|| 12&micro;L|| 30&micro;L
|-
|-
-
|Ptet-pT181 antisense(S+P) negative|| 0.5&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 7.5&micro;L|| 10&micro;L
+
|Ptet-pT181 antisense(Stel+Pstl) negative|| 0.5&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 7.5&micro;L|| 10&micro;L
|-
|-
-
|Pcon-RBS-lacZ&alpha;-DT(E+S)|| 7.1&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 3&micro;L|| 3&micro;L|| 14.9&micro;L|| 30&micro;L
+
|Pcon-RBS-lacZ&alpha;-DT(EcoRl+Stel)|| 7.1&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 3&micro;L|| 3&micro;L|| 14.9&micro;L|| 30&micro;L
|-
|-
-
|Pcon-RBS-lacZ&alpha;-DT(E+S) negative|| 0.4&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 7.6&micro;L|| 10&micro;L
+
|Pcon-RBS-lacZ&alpha;-DT(EcoRl+Stel) negative|| 0.4&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 7.6&micro;L|| 10&micro;L
|-
|-
-
|RBS-lysis2-DT(X+P)|| 22&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L|| 0&micro;L|| 30&micro;L
+
|RBS-lysis2-DT(Xbal+Pstl)|| 22&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L|| 0&micro;L|| 30&micro;L
|-
|-
-
|RBS-lysis2-DT(X+P) negative|| 1.4&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 6.6&micro;L|| 10&micro;L
+
|RBS-lysis2-DT(Xbal+Pstl) negative|| 1.4&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 6.6&micro;L|| 10&micro;L
|}
|}
</div>
</div>

Revision as of 13:24, 24 September 2013

Contents

Sep 11

PCR

Tatsui

pKaiBC(200p)10*buffer KOD plusdNTPsMgSO4primer mix(F&R)KOD plusMilliQtotal
0.52.52.51.51.50.51625
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　60°C　68°C30
2min　10sec　30sec　30sec　
pKaiBC(400p)10*buffer KOD plusdNTPsMgSO4primer mix(F&R)KOD plusMilliQtotal
12.52.51.51.50.515.525
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　60°C　68°C30
2min　10sec　30sec　30sec　
pKaiBC(800p)10*buffer KOD plusdNTPsMgSO4primer mix(F&R)KOD plusMilliQtotal
22.52.51.51.50.514.525
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　60°C　68°C30
2min　10sec　30sec　30sec　
pKaiBC(1ng)10*buffer KOD plusdNTPsMgSO4primer mix(F&R)KOD plusMilliQtotal
2.52.52.51.51.50.51425
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　60°C　68°C30
2min　10sec　30sec　30sec　

colony PCR

Tatsui

Samplebase pair
9/9Pcon-lacZα(p3B4k5)712bp
9/9Pcon-spinach-DT(p3B4k5)605bp
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
5min30s　30s　42s　
Samplebase pair
Pbad\araC-RBS-luxI-DT
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
5min30s　30s　2min　
Samplebase pair
9/9Pcon+RBS-luxI-DT1079bp
9/10Pcon+sptamer12_1R-DT584bp
9/10DT+Pcon-pT181 attenuater 1781bp
9/10Fusion1 attenuater(p3B1C3)-1638bp
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
50sec　30sec　30sec　1min　

Restriction Enzyme Digestion

Nakamoto

DNAEcoRlXbalStelPstlbufferBSAMilliQTotal
pSB4K5(EcoRl+Stel)9.4µL 1µL 0µL 1µL 0µL 3µL 3µL 12.6µL30 µL
pSB4K5(EcoRl+Stel) negative0.5µL 0µL 0µL 0µL 0µL 1µL 1µL 7.5µL 10µL
RBS-lacZα-DT(EcoRl+Stel) 10µL 1µL 0µL 1µL 0µL 3µL 3µL 12µL 30µL
RBS-lacZα-DT(EcoRl+Stel) negative 0.5µL 0µL 0µL 0µL 0µL 1µL 1µL 7.5µL 10µL
Ptet-pT181 antisense(Stel+Pstl) 10.8µL 0µL 0µL 1µL 1µL 3µL 3µL 12µL 30µL
Ptet-pT181 antisense(Stel+Pstl) negative 0.5µL 0µL 0µL 0µL 0µL 1µL 1µL 7.5µL 10µL
Pcon-RBS-lacZα-DT(EcoRl+Stel) 7.1µL 1µL 0µL 1µL 0µL 3µL 3µL 14.9µL 30µL
Pcon-RBS-lacZα-DT(EcoRl+Stel) negative 0.4µL 0µL 0µL 0µL 0µL 1µL 1µL 7.6µL 10µL
RBS-lysis2-DT(Xbal+Pstl) 22µL 0µL 1µL 0µL 1µL 3µL 3µL 0µL 30µL
RBS-lysis2-DT(Xbal+Pstl) negative 1.4µL 0µL 0µL 0µL 0µL 1µL 1µL 6.6µL 10µL
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