PCR sequencing (again!)

Dave Knorr dak at apldbio.com
Fri Jul 22 15:16:32 EST 1994


In article <1994Jul22.054431.10018 at news.unige.ch> Mike Morris,
mike at divsun.unige.ch writes:
>  We are trying to sequence PCR products by strand-separation with a
>biotinylated primer and streptavidin beads. Our first attempt produced
>no single-stranded DNA at all. Urghh.
>  Does anybody have a feel for the critical steps - I reckon my beads
>might be responsible: they have been sitting in teh cold room for about
>two years. Can they "go off"?

I believe Dynal beads will last about 18 months, depending upon storage,
contamination, etc.  If you're not using their beads then I'm not sure
how long they'll last.  How long is your PCR product?  Efficiency tends
to drop off as size increases above 1 kb.  What method did you use to
separate the strands?  I like 0.1 M NaOH the best (2-4 min. at room
temp).  Although I have had poor luck trying to recover the released
strand, others tend to have excellent results.  Why don't you just
sequence the remaining (bound) strand?

Dave Knorr
dak at apldbio.com



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