Hi, I am trying to mutate my gene of interest by PCR and then to transfer in competent Cell DH10b , My primer have length of 26bp and TM 70.14C , I have set the TM of 55C for my primer in protocol, I transferred my vector with gene of interest to DH10b but there was no colony . I have repeated for 3rd time but the same result , please let me know how to overcome this problem ?
1,you want to mutate the gene, you should use the enzymes with high conservation, such as PFX or KOD, which reproduce DNA at 68.
2,Tm 70.14 how did you get it ? different softs show different TM. It does not matter. You can use lower Tm(66) in PCR with lower specificity. 26 bp is enough for the specificity.
3, the 3' should be C or G ,which can have high specificity.
1. You may add DMSO( 4%, 6% or more) to PCR reaction.
2. Please checking again your primers(must be 5'-Phosphorylation)
You must check that your PCR produced the mutated gene first. Check the proper ligation conditions and then compare transfection of wild versus mutated. Also include proper controls in your transfection experiment to be able to interpret the results properly.
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