Hi everyone,
I was wondering if anyone has a solution for the amplification of long GC-regions using PCR?
I want to isolate ~5kb long sequence from mouse genome that is unfortunately rich in GCs. I am using Phusion polymerase with a buffer suitable for GC-rich regions amplification (NEB) and I tried DMSO, slowdown PCR, lower/higher annealing temperatures (depending on DMSO). I'm getting products of ~2kb and some bigger products that don't seem specific. I want to try betain as an additive and also ethylene glycol and 1,2-propanediol, but I was wondering if there is some approach that I don't know about?
Thank you for your help.
This can work, but it's hard. First thing would be to upgrade from phusion to Q5 polymerase. It's made a big difference for me, for that type of reaction.
The secret GC-rich buffer almost certainly already contains betaine, DMSO et al. in unknown concentration. So if you want to optimize these components yourself, you should use standard buffer. It could be enough to use standard buffer and add varying concentrations of secret "GC enhancer", like 0-10 ul in 2 ul increments.
Finally design some nested primer pairs. Some pairs work much better than others, and there's no great way to predict this. Use software from primer vending companies to design them.
I agree with John Schloendorn's suggestions. In addition, in case you need the piece of DNA for producing a recombinant protein, you may consider having a synthetic gene synthesized with codon optimization. For a 5-kb gene, the cost will be important, but well worth the saving of time and trouble.
Try this one. We have good expieriences with that kit in routine
LA Taq DNA polymerase with GC buffers
as well as the NEB LR Kits
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