small fragment library construction

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Thu Sep 23 10:03:37 EST 1999


Hi all,
I´m collecting ideas on how to make a library of short DNA fragments in
an expression vector. I have a 2.7kb cDNA fragment that I want to split
into many overlapping fragments in the 30-80 base pair size range. For
cloning, it would be best to have a restriction site on both sides of
the fragment, although in principle I could also use blunt end cloning
if no other possibility should work. I would prefer to have a PCR-based
method rather than an enzymatic digestion with e.g. DNase or micrococcal
nuclease. Digesting with a combination of restriction enzymes (or CviJI)
is not an option because of a quite long, highly GC-rich sequence that
is either not cut at all (by RE having A or T in their recognition
sequence) or chopped up to too small fragments (with GC-enzymes).

All suggestions welcome,
Frank





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