Team:Freiburg/Notebook/Methoden

From 2012.igem.org

(Difference between revisions)
(→Second Extension PCR (Toolkit))
(→First Extension PCR (Toolkit))
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|0,25 µl
|0,25 µl
|-
|-
-
|Water, nuclease-free
+
|Primer forward
-
|up to 20 µl
+
|1,25 µl
|-
|-
-
|'''Total vloume'''
+
|Primer reverse
-
|'''20 µl'''
+
|1,25 µl
 +
|-
 +
|Template
 +
|0,25 µl
 +
|-
 +
|'''Total'''
 +
|25 µl
|}
|}
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<br/>

Revision as of 18:20, 25 September 2012






First Extension PCR (Toolkit)

This step is necessary to add the the necessary restriction sites to the Direpeats.
Pipette the following volumes into a PCR- tube:

Component Volume
Water, nuclease-free 15,75 µl
DMSO 0,75 µl
dNTPs 0,5 µl
Phusion Buffer 5,0 µl
Phusion Polymerase 0,25 µl
Primer forward 1,25 µl
Primer reverse 1,25 µl
Template 0,25 µl
Total 25 µl



Second Extension PCR (Toolkit)

This step is necessary to add enzyme- binding –sites to the outer restriction sites. Use the product of the first extension- PCR as template.
Pipette the following volumes into a PCR- tube:


PCR Purification

bla


Gel Run

bla


Ligation

bla


Colony PCR

bla




Mini-Prep

bla


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