More Cloning Big

Suresh Joseph josephs at
Fri Jun 20 13:55:07 EST 1997

I know everyone is sick of cloning big insert questions, but please
entertain me: We are cloning a 8kb PCR product (as well as a traditional
fragment in a parallel project) into 5kb vector.  Problem is, when we
screen by restriction digest we see either vector only (expected) or what
appears to be vector plus an insert about 3kb.  We use NEB ligase,
traditional heat shock transformation protocol using subcloning efficiency
DH5alpha (Gibco).  We also vary ratios of DNA, and ligate O.N. at 16C in
10ul volume. DNA is from Quiaex purification of excised bands. Any
suggestions would be helpful
Darren Boehning 
Dept. Pathology and Cell Biology
Thomas Jefferson University

By the way, we get plently of colonies; about 50% insert and 50% this
mysterious reproducible smaller product.  We have also tried CIAP to no

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