your experience with long range PCR - (Aug/30/2007 )

Hi, if anyone has consistently produced a variety of ~ 10 kb sized amplicons from human genomic DNA, could you share your experience here? I'm probably going to generate overlapping 10 kb products on a high throughput format for sequencing in the near future.

Eg.
-enzymes/ kits especially the newer solely proof-reading enzyme formulations such as iProof, Phusion and Stratagene Pfu series
-relative ease of optimization (or rather minimal optimization required)
-robustness
-cycling time

thanks,
jenny

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Hi Jenny,

As you know, be very cautious in your template selection. For example, don't expect to be able to amplify 10kb from paraffin...
I've used a kit from Takara to amplify 8kb and got nice results in terms of yield and fidelity.
One of my colleagues published a paper:

Long-range (17.7 kb) allele-specific polymerase chain reaction method for direct haplotyping of R117H and IVS-8 mutations of the cystic fibrosis transmembrane regulator gene.
Pont-Kingdon G, Jama M, Miller C, Millson A, Lyon E.
J Mol Diagn. 2004 Aug;6(3):264-70.

Check it out. It may provide you with what you need.

Good luck!

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My template source is from blood or lymphoblastoid cell line, so there should be relatively free of inhibitors.

-jenny

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hi

i use LA Taq from taqara, my DNA is human genomic (from blood), and i have very good results until 9kb following the instructions of the kit.
i want to go up to 30kb. i havent tryied yet.
if you need more info tell me.

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