I digested 5ug of pLentiv2 (12kb) with 1uL BamHI-HF (NEB) at 37C for 2 hours in a 20uL volume with no heat inactivation required. I ran the digested vector on a gel (lane 4), and detected the presence of 2 bands which is similar to the uncut vector (lane 2). In the previous digests, i normally see one band and if i saw another band, it would be faint and i usually just cut the top and proceed with ligation with no problems.
Do you believe the vector is cut and I can proceed with purification or is the lower band the supecoiled conformation due to inefficient digestion and I should repeat it? If so what do you suggest i do differently ?
Dear Fiyin,
I would not proceed. There is too much uncut DNA. I would just repeat the digest with less DNA.
Best,
Uwe
Hi Fiyin,
I think that 2h of digestion with an HF enzyme should be plenty to fully digest any amount of DNA. So my guess is that your BamHI is faulty.
I have recently switched all my restriction enzymes to the FastDigest versions from Thermo. So far, all my digests were 100% done and successful and only took a fraction of the regular time. While more expensive, it saved me a lot of time and hassle! This may be a consideration for you if you need to replace your enzyme.
Good luck!
-Jan