Team:Kyoto/Notebook1

From 2012.igem.org

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==August 2==
==August 2==
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'''Mutation of FT'''
 +
{|class="wikitable"
 +
|+Inverse PCR
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!10xBufer!!2mM dNTPs!!primer fwd!!primer rev!!template!!polymerase!!MilliQ!!Total
 +
|-
 +
|5||5||1.5||1.5||0.5||1||35.5||50
 +
|}
 +
94°C 2min, (98°C 10sec, 68°C 4min)x2cycles, 4°C Hold
 +
 +
{|class="wikitable"
 +
|+Dpn1 Digestion
 +
!PCR product!!Dpn1
 +
|-
 +
|50||2
 +
|}  37℃,1h incubate
 +
 +
{|class="wikitable"
 +
|+Self-ligation
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!product!!MilliQ!!Ligase!!T4 Kinase!!Total
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|-
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|2||7||5||1||15
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|}
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16°C, 1h incubate
 +
 +
{|class="wikitable"
 +
|+Transformation
 +
!competent cell!!DNA
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|-
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|20||2
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|}
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Cells were stored on ice for 30min. <br>
 +
After 42°C 60sec heat shock, cells were stored on ice for 2min.<br>
 +
Then cells were preincubated at 37°C for 1hr, plated to Kanamycin plate.

Revision as of 13:14, 21 August 2012

August 2

Mutation of FT

Inverse PCR
10xBufer2mM dNTPsprimer fwdprimer revtemplatepolymeraseMilliQTotal
551.51.50.5135.550

94°C 2min, (98°C 10sec, 68°C 4min)x2cycles, 4°C Hold

Dpn1 Digestion
PCR productDpn1
502
37℃,1h incubate
Self-ligation
productMilliQLigaseT4 KinaseTotal
275115

16°C, 1h incubate

Transformation
competent cellDNA
202

Cells were stored on ice for 30min.
After 42°C 60sec heat shock, cells were stored on ice for 2min.
Then cells were preincubated at 37°C for 1hr, plated to Kanamycin plate.

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