Is PCR efficiency higher if mutation is in the middle of primer?
Posted 19 June 2012 - 06:24 PM
Posted 21 June 2012 - 05:38 AM
Other way only half of your product will contain the mutation and the selection is difficult.
But as for efficiency of the primer with mismatch on 5' end, it will be actually better than the missmatch in the middle, see that it's a common practise to put restiction sites that don't match on 5' ends. The closer the mismatch is to the 3' the worse AFAIK, for the efficiency. But mismatches near 5' end could cause the whole 5' end to unbind and then reduce the specifity of the primer, that is I think the reason why mutagenesis mismatches should be in the middle (but mutagenesis primers are quite long). If you have the primer long enough on the 3' part, this may not be serious issue.
I never trust anything that can't be doubted.
Posted 21 June 2012 - 07:26 PM
I have done site directed mutagenesis before, but I don't know why I thought mutagenesis can be done with only one primer on a linear template, silly of me....You also mentioned that the chance of having mutation with only one mutagenesis primer is just half. It makes sense. However, I think I will go ahead with my design and just ligate my PCR product to pJet and then extract plasmid from several colonies and just check which one has the mutated product??! I hope I can select the right one !
But even if I don't select the right one, I will run site directed mutagenesis with the PCR product in pJet later.
Edited by Curtis, 21 June 2012 - 07:28 PM.
Posted 08 July 2012 - 08:41 PM
It goes like this:
Product 1 ------------------------- Product 2
==================== + ===================
F primer > -------------------------------------------<mutagenesis R primer
Posted 09 July 2012 - 01:18 AM
Hope you aren't colour blind...
Posted 10 July 2012 - 01:12 AM
I think i finally got a band at 6.7 kb today after oe-pcr. I am ligating it to pjet and will send for sequencing. hope that's the correct band. my only worry is that the more oe-pcr i do,the harder i can purify them after gel extrsction.
Posted 10 July 2012 - 01:03 PM
Posted 10 July 2012 - 06:55 PM
This time I kept my overlap length at 20 bp based on what pcrman suggested last time...I think the longer the overlap size, the higher the Tm. I mean it will be difficult for high Tm regions to bind together. I kept it at 61C this time, to match the reverse and forward primers too. I think that was the trick.