I'm working with a plasmid that contains a 5.1kb insert of HCMV DNA (total size is ~8kb). Initially, I attempted site-directed mutagenesis on the entire plasmid but I could not obtain a product. We have successfully amplified up to 1.5kb in the region using mutagenic primers inside the fragment paired with primers in the vector to substitute between 4 and 6 nucleotides at a time. However, when I try to amplify/mutate with primers that give a larger product (between 3 to 4kb) I am getting multiple, shorter bands with a decent amount of smearing. There are many mononucleotide repeats in the larger product which we believe are making the PCR fail. We've tried lowering annealing temp, increasing extension time, adding formamide, raising MgCl2 conc., and recently touchdown PCR... none of which have been successful. The polymerase I'm using is Q5 from NEB which works very well in all other aspects.Does anyone have a suggestion for what I could change or add to my PCR in order to make it work?
Thanks Andrei. I should have mentioned that I am engineering restriction sites at the same time as mutating the sequence to disrupt protein binding. The original idea was to cut with those enzymes to confirm that the mutation was present. Recently, I switched to the subcloning method and was successful with a 0.5kb amplicon that has restriction sites on both ends. However, I still have to get it to work with a 3kb region, which is difficult - this is the region with many mononucleotide repeats (sometimes over 10 of the same base in a row). So, that is my current challenge!
We just ordered Phusion HotStart and I'm trying it out now, but I will also try regular Phusion as you suggested :)
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