Genomic library construction troubleshooting

shifali chatrath via methods%40net.bio.net (by shifalich from rediffmail.com)
Wed Mar 4 04:31:00 EST 2009


  Can you consider trying TA cloning using Promega's pGEMT easy kit? I have used this for cloning my cDNA library. Promega claims cloning of fragments as big as 10Kb or so.


On Tue, 03 Mar 2009 Rosario Díaz-González wrote :
>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.
>
>Rose
>
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Shifali Chatrath
Graduate Student
Protein science Lab
Dept. of Biological sciences
National University of Singapore
Singapore
+65-65161210
+65-96393449


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