Team:TU Darmstadt/Protocols/mutagenic PCR

From 2012.igem.org

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===Mixtures===
===Mixtures===
-
'''Reaction mix'''
 
-
5x Phusion HF buffer 5µL
+
{| class="wikitable"
-
dNTP´s 1µL
+
!Reaction mix !! Volume/Mass
-
DMSO 1,5 µL
+
|-
-
DNA 5,25,50 ng
+
|5x Phusion HF buffer || 5µL
-
Primer fwd 125 ng
+
|-
-
Primer rev 125 ng
+
|dNTP´s || 1µL
-
Phusion-Polymerase 0,5 µL
+
|-
-
H2O ad 50 µL
+
|DMSO || 1,5 µL
 +
|-
 +
|DNA || 5,25,50 ng
 +
|-
 +
|Primer fwd || 125 ng
 +
|-
 +
|Primer rev || 125 ng
 +
|-
 +
|Phusion-Polymerase || 0,5 µL
 +
|-
 +
|H2O ad || 50 µL
 +
|}
===PCR Program===
===PCR Program===

Revision as of 13:57, 24 September 2012

Contents

Mutagenic PCR

This site-directed mutagenesis is used to change particular base pairs in a piece of DNA.

Working principle

Figure 1.

The basic procedure requires the synthesis of a short DNA primer. This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so it can hybridize with the DNA in the gene of interest. The mutation may be a single base change (a point mutation), multiple base changes, deletion or insertion. The single-stranded primer is then extended using a DNA polymerase, which copies the rest of the gene. The copied gene contains the mutated site, and is then introduced into a host cell as a vector and cloned. Finally, mutants are selected.

Primers

primers designed for this PCR:

Mixtures

Reaction mix Volume/Mass
5x Phusion HF buffer 5µL
dNTP´s 1µL
DMSO 1,5 µL
DNA 5,25,50 ng
Primer fwd 125 ng
Primer rev 125 ng
Phusion-Polymerase 0,5 µL
H2O ad 50 µL

PCR Program

Retrieved from "http://2012.igem.org/Team:TU_Darmstadt/Protocols/mutagenic_PCR"