subcloning 9kb fragment

[yw Lee]

Hi netters,

I have some troubles concerning subcloning of a DNA fragment. A 130kb
bacterial artificial chromosome (BAC) clone was digested with XhoI,
fractionated on 0.7% LMP agarose gel in TAE, and a 9 kb fragment was
excised. The DNA was purified by QIAquick gel extraction kit (Qiagen)
and the resulting product quantified on gel, which was found to be about

10-15 ng. The 9kb fragment was ligated with approximately the same
amount of XhoI-digested pBluescript vector (SAP-treated) and the whole
20ul ligation mix was used for transformation into DH5alpha following
standard protocol. Unfortunately, the same procedures were done twice
and not even a single colony appear on the plate. I really have no idea
what has gone wrong. I did check the transformation efficiency of the
competent cells and found OK. I was wondering if my starting materials
were too scarce and would in-gel ligation help? If yes, please send me a

protocol. Any other input is very much appreciated. Thanks in advance.

yw Lee

email: ywlee at zmnh.uni-hamburg.de