Hi all!
I am doing site-directed mutagenesis to implement SNPs in a vector. I have successfully gotten all my single mutations and am now moving into creating haplotypes, so for example I'll take a construct that has L254P and then do S481G to make the genotype L254P S481G. I have not been getting colonies, and I really have a hard time believing one base change would reduce the mutagenesis efficiency from getting plenty of colonies to no colonies. I did have successful mutagenesis occur this time around with the same reagents. Would you guys give it another go with the same primer set, or revisit the primers?
Thanks,
Claire
If you used the same primers pair to create the same mutation in another plasmid, they should totally work, unless the new plasmid is much longer and you are not amplifying the entire plasmid sequence, and the plasmid is unfinished and unable to close itself and transform E. coli.
Also, are you 100% sure that all the reagents are really the same? One critical point is the E. coli strain you are using. It should have a high transformation efficiency. Maybe you could skip that genotype and try to do another, so you can test whether the reagents are really ok.
Good Luck!
Judith
It sounds like the problem is your transformation (or lack there of) and not your primers. Do you get colonies from any of your controls?
Judith Noda Katie A Burnette Hey guys! I don't think it is the cells as I had success with several constructs and used the same aliquots of competents. I use agilent quikchange so it's their cell line specifically for mutagenesis and are high efficiency. Vector is the same size, and I give 30 seconds longer than recommended a cycle and do 35 cycles.
OK, sounds like you've done the controls to rule out issues with your cells.
Try running out some of your plasmid on a gel to make sure it's the size & concentration you want.
Also, double-check that your SNP isn't in an essential gene (e.g. antibiotic resistance).
Good luck!
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