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Triple ligation not working

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#1 mithilesh



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Posted 29 December 2009 - 04:44 AM

Hi all,
I am trying to ligate insert 1 ( Nco1-Sac1, 2180 bp), insert2 (Sac1-Sph1, 335 bp) to a vector cut at Nco1-Sph1 sites ( 3.5 Kb). Initially, after ligation I used to get a band of around 6Kb but after transformation none of the colony screened show the presence of the insert in the plasmid DNA.
I treated the vector backbone with CIP and purified . Tried to religate the inserts with CIP treated vector but no ligation was observed on gel and no colony was observed after transformation.
Could anybody suggest anything ?

#2 jah



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Posted 29 December 2009 - 10:13 AM

Triple ligations are tough, but here are some tips I've picked up....

- Heat-inactivate CIP or gel-purify the dephosphorylated vector; I had a lot of problems with CIP and SAP carryover (even with heat-inactivation), so I've since switched to a thermo-sensitiveAP which helped a lot.
- I add ATP (2x) to the ligation, and incubate o/n at 8'C.
-Be gentle when mixing
-include a controls with an intact vector as a control, that way, you know whether the ligation or the transformation is the problem

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