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isolating 40bp DNA for recombination


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#1 donny

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Posted 27 December 2010 - 06:03 AM

I'm attempting to recombine mutants of a 3kb gene and am thinking of using Stemmer's method (ie DNaseI digestion, primerless PCR then PCR amplification). I found a paper that digests DNA fully using Mn2+ and DNaseI to ~40bp fragments. They purified the fragments with spin column. I'm just wondering if there are protocols to purify DNA of such small molecular weight without spin columns since that would add extra cost to my project. Are there any other recombination protocols? I've tried StEP PCR but am getting mostly smears and low yield, probably due to the size of the gene.

#2 BioMiha

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Posted 27 December 2010 - 08:00 AM

You can try the old EtOH precipitation. The yield bellow 100 bp is low, but enough perhaps for PCR. Otherwise even the commonly used spin columns won't do because most are not useful for fragments smaller than 100 bp. I used the Millipore YM-3 columns.




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