I have a 22kb backbone that needs to use Fse1 (NEB) and Kfl1 (Thermo) to cut to(21+kb and 700+bp bands). Still, the 2 enzymes come from different companies and their buffers are not the same(rcut-smart vs. fastdigest10 buffer), No matter whether I digested them together or separately, I couldn't get the right band, so I guess Fse1 didn't play a role ( I have already did the dam–/dcm– Competent E. coli C2925 transformation firstly), but Fse1 still can not cut my plasmid... So what should I do, try HST04 dam-/dcmCompetent Cells (form TAKARA)??? or redesign the vector?
Have you sequenced your plasmid to verify that the cut site has not mutated?
I would suggest you do each single digest first to see if both enzymes actually cut (or not). There are two things to consider, first one enzyme might not be cutting (so you need to confirm). Secondly if both enzymes are cutting fine but you are not seeing the right size bands, then maybe the plasmid is not correct.
Thank You for your suggestions. I will continue to discuss this with my doctoral advisor. By the way, I can not get the correct max prep DNA when I use the C2925H, but 6/36 mini DNA is right, so I have to use the Mini DNA...
but its concentration is insufficient, so I ordered SCS110 and HST04...and I will try them.
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