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Why don't get the desired ligation - (Oct/11/2004 )

I recently setup a ligation rxn with the insert and vector doule digested.
After incubated at 22 degree for 1hour (Fermentas protocol), I run the gel to see if the insert has been ligated. The gel shows a smear so I can't determine if the ligation is successful?
So I try the transformation( about 30-40 colonies grow in the LB plate) and purify the plasmid using Qiagen kits. After purification I single digest the extracted plasmid and run the gel to estimate the length of the lineared plasmid.
The gel shows a 8K band.( the original vector has 4k bps) So my insert doesn't ligate into the vector.
Could any one tell me how I can increase the ligation efficiency which means increase the chance of the insert and vector ligation instead of vector and vector ligation?
Thank you very much for your kind help!



Hi netnus,

I would use two enzymes which can release the insert, then run a gel. In this way I will definitely know if there is an insert in the plasmid. If you use one enzyme to linearize your plasmid, how about if there are still supercoiled plasmid whose migration in the gel is hard to predict. It is unlikely that all your colonies are plasmid-plasmid dimer.