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How to prepare double-stranded oligonucleotides?


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#1 wliu

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Posted 23 June 2004 - 04:10 AM

Dear all,

Anybody knows how to prepare a double-strand DNA fragment from synthesized oligonucleotides? and how to purify it to get rid of unbound single-strand oligo? Thanks in advance

#2 mich_ard

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Posted 23 June 2004 - 06:09 AM

hi there

how are you?

regarding how to purify dsDNA from synthesized oligos, i think it's pretty easy. i did it a couple of times for making adaptors. the primers i used were ~30 bases, add equal molar together, boiling in beaker, and then take off the beaker to your bench let it cool down to room temperature, it usually takes 30-45 min (i like to use 500 ml size beaker, so that it takes some time to cool down to room temperature). I didn't purify it from ss oligos, but if it's necessary you can run a 1.5-2% agarose gel to separate them.

hope this helps,

mike

#3 kant0008

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Posted 27 June 2004 - 04:54 PM

Hi!
My method's pretty similar. I add equimolar amounts of both oligos, but if you dissolve oligos in water, you have to then add STE buffer (I find as high as 90% STE total volume is good). Then heat at 95 for about 15 mins, and allow to cool slowly to room temperature- either use a heating block, and take the metal block off onto the bench, or use a pcr cycler (gradually cool). As far as purification, run on gel and purify. In theory shouldn't matter, as ssDNA will not be incorporated into anything you're working with, such as plasmids

#4 wliu

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Posted 28 June 2004 - 03:48 AM

Thanks for these suggestions. I still have some confusions needed to make clear. How do I know the efficiency of double-strand DNA preparation after heating and annealing? Anybody having a simple protocol to quantitatively detect double and single strand oligos will be highly appreciated.

Wei




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