Ligation problem-help!

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saraarasus
saraarasus's picture
Ligation problem-help!

Hi all,
I am having  problem with DNA ligation. I am trying to ligate the 375bp insert with xma1 and bamh1 site and vector with the same sites. After digestion for 2hrs at 37, I treated the vector with 1ul of CIP for 30 min and gel purify the digestet fragment (see the attached file) and vector with qiagene  quick gel extraction kit.
Then I did the ligation with quick ligation kit from biolab, I have tried 15min, 2hr and O/N at RT.
I have tride 5o ng of vector with insert to vector ration of 3/1,5/1 and 10/1 and I did not get even a single colony.
I have also tried  ethanol percipitation of ligation products and disolved the pallet in water and used all of it for transformation but still no colonies.

It is really frustrating and I do appreciate any suggestions.
Thanks,
Mitra