An easy solution would be to create multiple sets of primers and amplify regions and then stitch them together or do some overlapping extension PCR. This should be a viable option using Taq and splitting the DNA into 3-4 fragments.
On Tuesday, March 19, 2013 1:07:40 PM UTC-5, Mega wrote:
Hi everyone,I soon need to do a PCR of a ~10 kbp fragment.I asked in every lab of my university if they had some proofreading polymerase (like Pfu). Because the error rate of Taq polymerase is somewhat like 1:9000, it won't work with > 3 kbp.Buying one myself may not be an option, because I was told that those are remarkably expensive... (Althought, here it seems quite affordable... http://www.thermoscientificbio.com/pcr- 60€ for 100 units )enzymes-master-mixes-and- reagents/pfu-dna-polymerase/ Of course, I'd pay the mailing fee, and a little compensation. Or tade it for one of my plasmids. pVIB, pGreenII-0049, p35S-GFP. Have acces to pTracer-CMV2, so I could possibly share also this one.Just shoot me a mail if interested ;)
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