Trof, on 30 July 2012 - 03:04 AM, said:
You use 12 cycles as mentioned in the protocol, that may be not enough to see on a gel, so you can't be completely sure that transformation failed due to lack of product. You can test if your primers really work in these conditions (or you can optimise them) by increasing the number of cycles to say 25-30, in which case you definitely should be able to see a product. If you don't want to waste Dpn, you maybe don't need to treat it, as 10 ng of the original template may not be visible (but put 10 ng of the plasmid in same volume on the gel too, just in case, you would then compare intensities, since your product would be indistingushable from a linear form of the original plasmid). You can't use this for transformation of course, but you can test your primers and conditions without wasting more competent cells. But you need to repeat PCR with Pfu, since other cheaper enzyme could require different conditions and the optimisation would be useless.
Still, there is quite a chance that your primers simply won't work, ever.
You have to understand that no one can tell you if your PCR mutagenesis should work, you have some calculated Tm, that's itself is an estimate, then conditions don't only rely on Tm alone, but on the sequence itself. Your primers may have bad sequence, secondary structures etc. Then, primers may be OK, but the plasmid itself may contain regions that don't amplify well, there can be many reasons why it doesn't work, and many of those are not even understood yet.
To be brief.. your conditions look fine as they are, but that doesn't mean anything, but it may actually require higher Ta, or lower Ta. Or it may require primer redesign. Or completely different approach as mentioned. You have to try, with the approach I mentioned above for example. There is no other way. Sometimes mutagenesis works on first try, sometimes it's pain in the ass.
What did you use to design your mutagenesis primers? Can you post the sequences? That may shed some light on quality of your primers, but it may probably only tell you if your primers seem bad (in that case redesigning would be strongly suggested) or OK (which doesn't tell you if they actually will work).
>sd arv1 codng
I have to design three mutations
primer set1(Tm= 72.6 oC
)- Forward: 5' CAATATACGAAAGCATATCGGCC
Reverse: 5' GGATTGTTGGTATTCAGTAACAAGG
primer set2(Tm= 72.5 oC)
- Forward: 5' GCATATCGGCACTTGTTACTGAT
Reverse: 5' GACAGTAGGGATTGTTGGTAA
primer set3(Tm= 71.9 oC)
- Forward: 5' CGGTATACGCCTAACAAATTC
Reverse: 5' GCATCTACAGGTTTCCCAG
underlined and in red are the mutation sites