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jlf 12-29-2010 05:27 AM

Purifiying 40bp DNA for recombination
 
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. Would EtOH precipitation work? Also, 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.

luisillo 01-05-2011 09:47 PM

Re: Purifiying 40bp DNA for recombination
 
Can you run your 40 bp fragment on a gel and purify it from it? You can use either agarose or polyacrilamide, but I would suggest the latter at 12% considering the size of your fragment.

I don't know how expensive are the gel purification kits in comparison with spin columns, but I suggest you check it since its likely that you'll have to purify that DNA in a better way than ethanol precipitation.

jlf 01-07-2011 07:46 AM

Re: Purifiying 40bp DNA for recombination
 
Based on the paper, DnaseI with Mn2+ digest DNA only down to 40bp and not much further, so the DNA mixture consist of almost exclusively 40bp fragments. Moreover, for my application, the size of the fragments is not critical since all I want is a variety of fragments for recombination. I just need to ensure DNase and Mn2+ are deactivated/removed so that they don't interfere with the subsequent PCR. I know Qiagen has a nucleotide removal kit that's suitable for my application. Are there any cheaper alternatives or brands?

luisillo 01-10-2011 08:39 PM

Re: Purifiying 40bp DNA for recombination
 
Ok. Can you do phenol:chloroform:isoamyl alcohol and chloroform:isoamyl alcohol washes to get rid of the enzyme?

Mn2+ ions could be chelated with an EDTA buffer. Why not resuspending the DNA pellet in TE (Tris-EDTA) buffer?

When done properly both methods work well for PCR purposes. The first is time consuming but both are money savers.


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