User:Maximilian Peters/site-directed-mutagenesis

Site-directed mutagenesis protocol

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

1. Primer design with Stratagene's primer design tool
2. Order both primers 5'-phosphorylated
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.

1. /
2. Primers were ordered from IDT DNA and TMs and primer-dimers were determined with Finnzymes' TM calculator (dimer detection sensitivity can be set to 4)
3. PCR reactions were performed with Phusion® High-Fidelity PCR Master Mix from Finnzymes using the standard protocol with the calculated TMs
4. PCR products were cut from the gel and purified with the NucleoSpin® Extract II kit from Macherey Nagel, elution volume 20 microL preheated to 70°C
5. Ligations were performed with Rapid DNA Ligation Kit from Fermentas, 15 μL elute, 4 μL Ligation buffer, 1 μL ligase, 1h at 22°C
6. 20 μL ligation reaction were used to heat-shock 200 μL of competent DH5α bacteria
7. Expected 20 to several 100 colonies, colonies were picked with a sterile tooth pick, grown overnight at 37°C and isolated with the PureYield™ Plasmid Miniprep System from Promega
8. Enzymes from NEB were used and the above mentioned kits

Comments

• I failed to amplify a vector >10 kb with Phusion, I therefore PCRed the gene of interest with a ~200 bp overhang on each end and cloned into the pJet1.2 vector. The generated vector was considerable smaller (7.5 kb) and could be used for the mutagenesis protocol. Perhaps other high-fidelity PCR enzymes might work with larger vectors.
• All colonies tested so far had only the desired mutation and no other mutation was introduced with Phusion