Hi guys I'm looking to use SDM to change a single residue in an enzyme I'm working with. I'm new to this so I thought I would ask advice. Is there a current protocol that works best or should i use the Agilent quickchange kits?
Hi....You can simply do PCR using your forward and reverse primers having point mutation using DNA polymerase enzymes. The choice of polymerase depends on the size of your plasmid (gene+vector). Prefer to use Phusion high fidelity polymerase for longer genes. Protocol of usage is provided along with the enzymes kits.
You can use different Kits.
http://kirschner.med.harvard.edu/files/protocols/Stratagene_quickchangepdf.pdf
http://www.genomics.agilent.com/en/Site-Directed-Mutagenesis/QuikChange-II/?cid=AG-PT-175&tabId=AG-PR-1161
https://www.agilent.com/cs/library/usermanuals/Public/200521.pdf
I agree with Nandini. My favorite protocol just involves amplifying the whole plasmid using non-overlapping phosphorylated primers containing point mutations with Phusion, gel extracting the product, then ligating and transforming. SDM isn't that complicated and in my opinion it's only worth ordering a kit if you won't use the enzymes for anything else.
I agree, I just do the mutation primers. I use this protocol for reference, which is from the provider of our enzimes https://www.nzytech.com/files/brochures/MB012_NZYMutagenesis%20kit.pdf?e6993b. From here, I take the primer specifications, the PCR mix and the PCR cicle. Do not forget to digest your DNA with DpnI enzime, according to the manufacturer recommendations. It is also important to transform your DNA into specific cell lines designed for this procedures (the most common being XL2)
Agree with the comments here already. I have performed a tremendous amount of mutagenesis and I've had the best experience with NEB's Q5 mutagenesis system: https://www.neb.com/products/e0554-q5-site-directed-mutagenesis-kit#Product%20Information
No need to buy the kit. You can hack it for cheap. Get the Q5 HF polymerase and primers for the initial PCR and make your own KLD mix (1 uL PNK, 1 uL NEB quick ligase, 1 uL DpnI, 1 uL PCR reaction product, 6 uL nuclease free water). Incubate for five minutes at room temp, then transform competent cells.
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