I am trying to prepare the vector for future cloning but bothered by high background. Below is my experiment setup.
The vector is pFBDM, around 5.7Kb in size. I choose two restriction sites which span 45bp apart. One site is for XmaI , the other is for KpnI.
I purchase two enzymes from NEB as fresh stock.
I use 1ug of plasmid ( purified by qiagen mini-spin kit ) as starting material. Double digestion was setup at 37 degC, the solution volume is 50uL. 1uL (20U) of restriction enzymes were used in the digestion reaction. I digest the plasmid for 2 hrs. After the digestion, the mix was purified with Qiagen PCR clean up kit under the guidance from manufacturer that it can remove DNA under 100bp. The choice was under the consideration that UV exposure can markedly decrease the transformation efficiency and we don't have long-wavelength UV box in our lab. Another consideration is we plan to clone a fragment of 3.8Kb in size, so the efficiency is of upmost importance to us. After digestion, I test with gel; it looks good.
I tested a aliquot of mixture for transformation. Surprisingly, I got hundreds of colonies ( DH5-alpha, efficiency around 5x10^7- 10^8 CFU/ug plasmid ). They are all uncut plasmids.
I think the problems are 1. incomplete digestion of the plasmid 2. the kit did not remove the digested small fragments efficiently, so self-ligation occurs.
Any suggestions for that? How to avoid gel-extraction? I know gel extraction might solve this issue, but I encountered marked decrease in efficiency. I had tested that.
Thanks a lot for your opinions.