Team:Kyoto/Notebook1

From 2012.igem.org

(Difference between revisions)
(Created page with "==August 2==")
(August 2)
Line 1: Line 1:
==August 2==
==August 2==
 +
'''Mutation of FT'''
 +
{|class="wikitable"
 +
|+Inverse PCR
 +
!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
 +
!product!!MilliQ!!Ligase!!T4 Kinase!!Total
 +
|-
 +
|2||7||5||1||15
 +
|}
 +
16°C, 1h incubate
 +
 +
{|class="wikitable"
 +
|+Transformation
 +
!competent cell!!DNA
 +
|-
 +
|20||2
 +
|}
 +
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.