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.
• The protocol is a modification of the Finnzymes Phusion™ Site-Directed Mutagenesis Kit, http://www.neb.com/nebecomm/manualFiles/manualF-541.pdf
• The plasmid with the gene of interest is PCR amplified with two primers. One primer carries the mutation and the second primer starts exactly one base before the other primer. The resulting PCR product is the linearized vector with a point mutation. The PCR products is then gel purified, ligated and used for transforming bacteria. The gel purification step eliminates contamination with the wild type vector. The resulting vector can be either directly used or the gene of interest is isolated either by PCR or restriction enzymes and cloned back into the original vector.
• If you have any questions or problems with this protocol, feel free to contact the author.

1. Design the forward primer with Stratagene's primer design tool (QuikChange II) and the reverse primer manually. The reverse primer has to start exactly one base before the forward primer and the TM should be higher than 72°C.
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 50 μL 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. Isolate 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. https://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Tool&SubPageType=ToolQCPD&PageID=15
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. Expect 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 it into the pJet1.2 vector. The generated vector was considerable smaller (7.5 kb) and could be used for the mutagenesis protocol.
  

The KaPa HotStart ready mix amplified plasmids >10 kb without any problems, however after transforming the ligation product there were far more recombination products, which were never observed with Phusion.

• All colonies tested so far had only the desired mutation and no other mutation was introduced with Phusion, as well as with KaPa.