Long PCR for amplification from plasmid - (Oct/05/2005 )
Well, I am trying to amplify a region from pIGCN21 plasmid to further do "recombineering". My primers are 63ntds in lentgh and 3` end is complimentary to the plasmid while 5` end is homolog to the target genomic BAC sequence. Their Tm is 72degrees for both. I have tried to amplify the cassette from the plasmid using Expand PCR kit from Roche. Did several dilutions for MgCl, tried different temperature profiles buy all i'm getting is primer bands. Any suggestions? Thanx
PS: I haven't checked for secondary structures within the primers. where can i do that?
what do you mean by 'primer bands'?
are you seeing primer-dimers? then you need to check your oligos better because if they have a high affinity for one another, changing all the other components won't get you anywhere
I've used the PCRx Enhancer System (protocol here) from Invitrogen for tough PCRs, and it has worked wonderfully. It's probably just DMSO, but whatever it is, it's cheap, and has allowed me to PCR up things that failed over and over using the usual way...
Their Tm is high, probably because they are long primers. Determine the Tm for just the annealing 15-20 bp or so and then try PCR again maybe?
I've posted this before, but I strongly advise sequencing the coding regions of the PCR product that you will be using for downstream applications. There is an error rate in all these polymerases (the proofreading ones are definitely lower) but it is better to know that what you have is correct rather than waste a year of your life working with something with a premature stop codon or mutation. If you want to be cheap and trust your product go for it, but it may make you sleep better at night!
It is DMSO and Betaine.
Most of the PCR enhancement systems out there are merely these two compounds. Promega researched this six years ago and reached this conclusion through NMR spectroscopy.
I'd try DMSO 1.3%, Betaine 1.3 M final concentration (recommended by wustl research). That should help...DMSO and Betaine are ridiculously cheap.