Genomic library construction troubleshooting

[Rosario Díaz - González]

Thanks all for the tips. Unfortunately, I can't not ride off of CIAP since my IP is really fond of with that enzyme, don't tell him about trying SAP. The same happens with the EtOH ppt, he told me to do it that way.

The genome is from a protozoan, and the E. coli strain we're using for the cloning is DH5alpha. The thing is that dephosphorylation works fine, it doesn't religate again but the few colonies i get are w/o any kind of insert, in fact it doesn't seem even to self-ligate: i even tried to pre-warm the vector-insert mix @ 68º, 3 min, to prevent the spontaneous self-ligation, but no better result.

The control reaction (dephosphorylated vector + ligase) plate is empty, and the ligation plates have about 20 colonies... I'm thinking it might be the insert,but don't get what neither why!

About the TA cloning, I considered it but how i got it into pGEM, with A-coiling? And wouldn't it be more difficult to get into the double-hybrid peyd vector with two different enzymes -those I need to use to get back the insert- when with one enzyme I'm going crazy? Maybe I can try, since I would jump over the CIAP :-)

Thanks again to everybody, guys.

On 4 Mar 2009 09:31:00 -0000, shifali  chatrath wrote
>   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
> >