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short dsDNA purification

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short dsDNA purification

Postby dldavide » Wed Jul 04, 2007 1:37 pm

Dear all,

I'm trying to construct a library of short dsDNA (average lenght 60bp), therefore I need a quantitative purification protocol to purify short dsDNA after PCR and/or restriction reactions. I tried both commercial kits and the traditional EtOH purification without success, the yield is too low (i lose up to the 80% of the input). Has anyone ever experience such problem? Has anyone a smart tip?

Thank you,
Davide
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Postby blcr11 » Wed Jul 18, 2007 4:23 pm

Not sure why you want to make 60-mers or shorter by pcr. Wouldn't it just be simpler to synthesize oligos and then purify by hplc? Of course, maybe I just don't understand what you're tying to do. If it is some sort of mutagenesis experiment, you can build that into the design of the oligos.
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dsDNA purification

Postby dldavide » Wed Jul 18, 2007 5:37 pm

Hi,

I'm constructing a completely random DNA library for in vitro transcription. I need to convert the ssOLIGO to a double-stranded form prior to the cloning into the receinving vector. Since the DNA library is random i cannot design a forward and reverse oligos, thus i use pcr to convert a ssOLIGO to a dsDNA. I'm unaware of any alternative method to do it, do you have suggestion?
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Postby blcr11 » Wed Jul 18, 2007 7:47 pm

It sounds like what you want to do, then, is something like saturation mutagenesis of a 60 base sequence of DNA, if I understand you correctly. Here are three papers who have done something similar.

http://www.pubmedcentral.nih.gov/articl ... tid=373423

http://jb.asm.org/cgi/content/full/181/8/2513

http://www.biologicalprocedures.com/bpo ... 24/m24.pdf

I don't know if any of them will help or not. I haven't looked at them that carefully, so I'm not neccessarily endorsing any of them, but perhaps they will give you some ideas. Another thought would be to design the oligos to have sticky ends for restriction sites so they hybridize in as ssDNA. That will create a 60 base gap that can either be filled in with polymerase (and spare you the trouble of making the dsDNA as a short piece of DNA that needs to be isolated), or, if you're feeling lucky, can just be transfected directly--gap and all. With the latter "technique," most likely nature will fill in the gap, but you leave yourself open to deletions or funny recombination events--it's faster, but maybe not better. Still possible I don't fully understand the problem, but that's my 2 cents, anyway.
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Postby dldavide » Thu Jul 19, 2007 9:01 am

Hi,

Thank you so much. I'll have a look to the papers. Concerning the ssDNA hybridisation, we are already trying but it seems to not work properly. Have you any reference about it?

Cheers,
Davide
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Postby blcr11 » Thu Jul 19, 2007 11:48 am

I don't, sorry. I do LIC cloning and I was thinking of something along those lines, but LIC uses dsDNA with suitable sticky ends prepared by T4 DNA polymerase's error-correcting activity. I suspect the problem with using long stretches of ssDNA is the potential for hairpin formation, especially near the ends, that might interfere with hybridization. I would use sites with as long an overhang as I can get and then cut the annealling temperature as close as possible--if the Tm is, say 60, anneal at 58, for example. With LIC cloning, you don't need to denature; room temp annealing is fine, but that is an overhang of something like 15-20 bases. You might have to denature first to make sure your oligos are as linear as possible. On the other hand, if you've been denaturing and it hasn't worked, try once without denaturing and see what happens-or try annealing at room temperature with or without denaturation. Don't think I've got any brilliant ideas to try, though.
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