For some unknown reason we had very poor success rates with the pCAGGS mammalian expression vector. We want to subclone our gene of interest into a vector, perform Quikchange and then clone it back into pCAGGS. Can anyone help?
I have had no luck performing Quikchange in pCAGGS. Once the gene was subcloned into pcDNA all of the mutagenesis worked, but I do not get good expression. When I subclone back into pCAGGS I get great expression. The process is a pain, but it works this way.
We have the same problems with quick changes in pCAGGS - a good alternative could be to do a two step mutagenesis PCR and then directly Gibson ligate the PCR product it back into pCAGGS, which saves you the trouble of sub cloning. I'm trying this right now.
I don't know the pCAGGS vector but in general the size of the vector is a crucial factor for the Quikchange methodology since the whole vector has to be amplified in the process. So small vectors like pUC19 or a TOPO vector are better suited for the mutagenesis PCR.
An alternative to tedious subcloning might be to use the Gateway system: You create the entry clone with you gene of interest, use this one to introduce all mutations of interest and then recombine the mutants into your expression vector. The pCAGGS or any other vector can be made Gateway compatible with a conversion kit that introduces the Gateway cassette into the multiple cloning site:
https://www.lifetechnologies.com/order/catalog/product/11828029
A second alternative to Quikchange is a relatively new mutagenesis kit from NEB, which works really well in my hands. In contrast to Quikchange it does not use overlapping mutagenesis primers but primers, which "meet" at their 5'-ends. This has the big advantage that besides introducing mutations you can very easily introduce bigger deletions or insertions (which is not possible with Quikchange). It contains a mix of enzymes which facilitate amplification, phosphorylation and ligation of the mutants. Maybe it's worth a try whether pCAGGS constructs can be mutated with this kit. Would be the fastest and easiest I guess.
https://www.neb.com/products/e0554-q5-site-directed-mutagenesis-kit
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