large-scale prep of ssDNA

Duke Groebe drg at prophet.pharm.pitt.edu
Wed Mar 6 10:18:41 EST 1996


In article <4hfm66$8mb at charm.magnus.acs.ohio-state.edu>,
yulee at magnus.acs.ohio-state.edu (Yuandan Lee) wrote:
> 
> Dear netters,
> 
> I'm looking for a method to prepare a large amount of single-strand DNA 
> (several hundred micrograms). I have subcloned the DNA fragment into the 
> Bluescript vector. The protocol I have in hand is basically for the sequence 
> reaction, therefore will give very small amount of ssDNA. Your help world be 
> appreciated.
> 
> Dan Lee
> lee.634 at osu.edu
> 614-261-1467


I'm guessing that your DNA is too long to be made synthetically.  One
thought I had is to use large-scale PCR to amplify your DNA but use a
biotinylated 3'-primer.  This way, you can denature the amplified product
and run it over an avidin-agarose column to remove the unwanted
complementary strand.  See, for example, the methods section in Bock et al.
Nature 355, 564-566 (1992).

Good luck,

Duke

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