CIP is the problem?

Chunxin Wang cxwang at wam.umd.edu
Mon Jan 10 15:25:29 EST 2000


Hi, all:

I failed to make a cosmid library from a BAC clone. would you give me some
suggestion?

To subclone the BAC of my interest, I tried to construct a cosmid
library. The vector used is pCLD04541 and clonging site is Cla I. The BAC
DNA was partially digested by Taq I, then the 20-25kb fragments was 
recovered from the agarose gel and ligated into the above vector. 3 ul of
ligation was used for packaging by Gigapck III XL Packaging Extract
(Stratagene)

Unfortunately, I did three times and failed to get even one clone. The
lambda DNA control shows that the packaging extract is no problem. Then I
used the ligation products of a YAC clone(which has been
successfully packaged) as another control and it works very well, implying
that my handling for package is ok. Therefore, the only thing should be
suspicious is the quanlity of the ligated DNA.

To purify the vector DNA, I used the agarose gel to recover the digested
vector and then purify with the Bio-Rad Prep-A-Gene kit. It still didn't
work and I always got nothing, zero colony even titering without dilution.

Then someone suggested the excess CIP might be the problem because I used
one ul of the undiluted CIP(10u/ul) from Biolabs for dephosphorylation. I
don't believe it because the CIP was finally inactivated by heat and
extracted by phenol. It should have nothing to do with the packaging.

Any suggestion is higly appreciated.

Black

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