If you just need DCM- DNA, but DAM methylation doesn't matter I would recommend DC10B if you can get it, as long as making your own competent cells isn't an issue.
https://ecoliwiki.org/colipedia/index.php/DC10B
Dcm doesn't affect much (other than methylating obviously) but Dam performs multiple important functions in E. coli, and Dam- strains tend to be kinda sickly and have a higher mutation rate. If I remember correctly they also need to be recA+ or else they die.
If you need totally unmethylated DNA, then SCS110 or C2925H seem like the best options to me because they're endA-. C292H is chloramphenicol resistant which limits your options for vectors that can be transformed into it. SCS110 contains the F plasmid but the notation doesn't make it clear if it's integrated into the chromosome or replicates separately. Not sure if that's a problem for your purposes or not.
Dcm only methylates at the sequences "CCAGG" or "CCTGG". Do either of these sequences actually overlap with the FseI recognition site? This would look like 5' CCAGGCCGGCC 3' or 5' GGCCGGCCTGG 3'. (FseI site bolded). If neither of those sequences is present, it's not being Dcm methylated even in a strain that's Dcm+.
As plasmid extracted from C2925H is not working, my suspicion is this has nothing to do with Dcm methylation at all. It could be the result of your restriction enzymes going bad, the enzymes being used in suboptimal conditions (wrong buffer or wrong incubation temperature), or that the sequence you were provided for this plasmid is just incorrect.
Thank you for your patience and care. As I am a physician in China, I have forgotten a lot of basic medical knowledge, so I can not discuss and communicate with my doctoral advisor on professional issues related to cloning. I am sending you the sequences of plasmid, maybe you can help me have a look.
I have a 22kb backbone that needs to use Fse1 (NEB, I always store it at -80 degree-C) and Kfl1 (Thermo) to cut to(21+kb and 700+bp bands). Still, the 2 enzymes (both of them are fresh and new) 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), Fse1 still can not cut my plasmid...but they can cut my insert DNA to the right bands that means the enzymes are good.
Looking at it, it does look like there is a CCAGG site that overlaps with the FseI site in that plasmid. Did you cut your insert and plasmid (extracted from C2925) at the same time? Just trying to rule out inactivation of the enzymes between freeze-thaw cycles, because NEB makes it sound like a pretty unstable enzyme.
At this point though, I would definitely get that part of the plasmid Sanger sequenced, just in case there isn't actually an FseI site there. I've spent a lot of time getting frustrated with cloning only to find out the plasmid map I was given was never validated via sequencing and the restriction sites I was trying to cut did not exist.
yes, at the same time, but only the insert plasmid can be cut to the right bands, the backbone did not work, I've advised my boss to send it to be resequenced...to make sure the Fse1 site is not mutated...by the way,A strange thing happened, the backbone plasmid that was digested by Fse1 and Kfl1, after one night at 37 degrees Celsius, I took 10ul of loading gel, and it couldn't be cut(1 band), but the rest I left one night, and after running the gel again, the backbone DNA could be cut(3 bands), but the size and number were wrong......should be 2 bands not 3...? thank U
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