5' end cDNA cloning
GLYCOBIOLOGISTS OF BUFFALO
camlau2 at ubvms.cc.buffalo.edu
Tue Jul 20 14:33:00 EST 1993
Has anyone out there used Clonetech's new AmpliFINDER RACE kit to
clone cDNA 5' ends? (It uses ligation of an oligo to the 5' end rather
than the troublesome homopolymeric tailing of the more traditional
procedure) Or has anyone done it without a kit? I know someone who's having
no luck doing it by himself (sans kit) using the same strategy, etc.. I've
used BRL's 5'RACE kit ( which involves tailing) after several of us failed to
make it happen on our own.
Over the years we've screened a number of commercial libraries, tried
RACE, and I've recently made my own library, only to find that the clones don't
extend far enough. I'm beginning to think this 5' end has some very stable
secondary structure or something, because no primer extension or cDNA synthesis
has ever resulted in anything near the full length product.
If this subject has been discussed to death, please forgive me. I've
been out of touch for a while (busy cloning!!) If you can offer any assistance,
I'd appreciate it.
Thanks in advance,
camlau2 at ubvms.cc.buffalo.edu attn:AVB
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