User:Maximilian Peters/site-directed-mutagenesis

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(New page: ==Site-directed mutagenesis protocol== This protocol does not require any special enzymes or kits, only a regular PCR block and equipment to handle DNA. 1. Primer design with Strategene'...)
Line 3: Line 3:
This protocol does not require any special enzymes or kits, only a regular PCR block and equipment to handle DNA.
This protocol does not require any special enzymes or kits, only a regular PCR block and equipment to handle DNA.
-
1. Primer design with Strategene's primer design tool
+
1. Primer design with Strategene's primer design tool<br>
-
2. Order both primers 5'-phosphorylated
+
2. Order both primers 5'-phosphorylated<br><ref group="materials"></ref>
-
3. Perform a 20 microL PCR reaction to test the primers, if a strong band in the desired region is obtained repeat the PCR with 2-4 50x microL reactions
+
3. Perform a 20 microL PCR reaction to test the primers, if a strong band in the desired region is obtained repeat the PCR with 2-4 50x microL reactions<br>
-
4. Run the PCR product on an Agarose gel (1% or less) and isolate the desired band with a method of your choice
+
4. Run the PCR product on an Agarose gel (1% or less) and isolate the desired band with a method of your choice<br>
-
5.
+
5. Ligate the purified PCR product<br>
 +
6. Transform your bacteria of choice with the ligated PCR product from step 5<br>
 +
7. Isolated three (or more) colonies and confirm the mutation by sequencing<br>
 +
Optional but recommended<br>
 +
8. Isolate the mutated insert with restriction enzymes and reintroduce it into the original vector to avoid background mutations introduced by PCR<br>
 +
 
 +
 
 +
===Materials===
 +
The following kits and enzymes were used for the protocol, but are not essential and any other kit or method might work as well.

Revision as of 10:17, 8 June 2011

Site-directed mutagenesis protocol

This protocol does not require any special enzymes or kits, only a regular PCR block and equipment to handle DNA.

1. Primer design with Strategene's primer design tool
2. Order both primers 5'-phosphorylated
<ref group="materials"></ref> 3. Perform a 20 microL PCR reaction to test the primers, if a strong band in the desired region is obtained repeat the PCR with 2-4 50x microL reactions
4. Run the PCR product on an Agarose gel (1% or less) and isolate the desired band with a method of your choice
5. Ligate the purified PCR product
6. Transform your bacteria of choice with the ligated PCR product from step 5
7. Isolated three (or more) colonies and confirm the mutation by sequencing
Optional but recommended
8. Isolate the mutated insert with restriction enzymes and reintroduce it into the original vector to avoid background mutations introduced by PCR


Materials

The following kits and enzymes were used for the protocol, but are not essential and any other kit or method might work as well.

Personal tools