I've cloned the gene for a target protein into a plasmid and, upon sequence verification, caught a single nucleotide missense gene mutation (L to P). Does anybody have a favorite way of dealing with such a problem? I know different techniques exist, but I'm hoping to hear pros and cons, etc. Thanks!
Do site-directed mutagenesis using Quikchange (from Agilent). If the plasmid is relatively small (i.e. shorter than 10 kb) it doesn't get much simpler or faster than that.
Thanks, Alejandro. I've been looking at a kit from NEB (Q5, I think?) that looks similar. I've used Agilent products in the past though, and been very happy with them. I appreciate the feedback!
You don't need any kit, what you need is a proofreading thermal DNA polymerase. Just following the protocol described in the publication http://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-8-91 and this protocol has been proved with high efficiency.
Thanks for the protocol, Huanting. This is similar to what my mentor was explaining to me.
1. You may order a G-block to replace mutant region by ordinary cloning.
2. Did you get only one clone? If you could get two clones with different mutations, you might recombine their parts lacking mutations.
Thanks, Michael. I did indeed get multiple clones (two, both with different mutations).
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