ss DNA from pcr?

chai_z at wehi.edu.au chai_z at wehi.edu.au
Wed Feb 10 01:23:11 EST 1993


In article <1l4cj4INNora at huon.itd.adelaide.edu.au>, rbonfigl at waite.adelaide.edu.au (Rod Bonfiglioli) writes:
> hello there,
> this is a repost as i didnt recieve any replies last time.  my system was down
> and i do not know if any one replied. if you replied to this question last time
> it was posted, could you please reply again, as i got no posts.  thanks in
> advance.
> I am looking for a pcr protocol to generate, or otherwise obtain large amounts
> of single stranded cDNA.  i have the m13 method, but i would prefer to use a pcr
> method if anyone has any good ideas.  i hope some of you people out there may
> have some ideas for me.....thanks in advance.......
> 
> rod
> --
> Rod Bonfiglioli, Waite Agricultural Research Institute,
> University of Adelaide, South Australia.
> e-mail rbonfigl at waite.adelaide.edu.au

A normal PCR should be carried out to produce good amount of dsDNA fragment
firstly. Phenol-chloroform, chloroform extraction are followed by saphacryl
column, or gel-purification of the dsDNA (to erase the BOTH PCR primers). Use
a big amount of the PCR fragment as template and only one primer to produce
ssDNA by PCR.

Altanatively, there are kits for solid-phase sequencing. Briefly, one of the
PCR primer is labelled with biotin (?) before pcr. The pcr products were then
denatured and the biotin binds to avidin (antibiotin?) on the surface of
magnetic sphare. After wash, only the strand labelled by biotin is retained,
that is ssDNA. It was claimed to give very good sequence, and to be a very
reliable method.

Further info. is available if you are interested.

Good Luck

Zhonglin Chai
Melbourne



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