Genomic library construction troubleshooting

Rosario Díaz - González via (by rdiazg from
Tue Mar 3 07:10:09 EST 2009

Hi everybody,

I'm working on a genomic library construction for 2-h, but I'm unable to get
any insert in my vector.

I'm working with pACT2 vector, digested with Bam HI and dephosphorylated with
CIAP (also tried with SAP). The insert is a partially Sau3A digested genomic
DNA, and ligated with different ratios (1:1, 1:3, 1:5). Both the vector and
the insert are agarose-cleaned by Qiaex, and the ligase is EtOH ppt.... but
all the few colonies I got are empty, w/o any insert in.

Anything I'm missing? Any tip or hint?

Thanks a lot in advance.


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