large-scale prep of ssDNA

[Michael Benedik]

In article <drg-060396101211 at ache.pharm.pitt.edu>
drg at prophet.pharm.pitt.edu (Duke Groebe) writes:

> > 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

If you don't mind having vector DNA attached, you might just subclone
it into M13. In my hands I get much higher (SS) DNA yields using M13
than using Bluescript or phagemids. You should be able to get hundred
microgram range from a liter prep.


Michael Benedik
Department of Biochemical Sciences
University of Houston
benedik at uh.edu