Hi everyone!
I have used 10U of SpeI (NEB or Promega) in restriction of P. aeruginosa plugs for PFGE, but we have now utilized the Thermo Scientific FastDigest BcuI restriction enzyme and the results are not good. The concentration is not showed in the label, only the volume of 50uL. Recommendation of 5uL did not work! All the strains presented the same "pattern" (or the absence of one).
Thiourea did not change the result.
Has someone experience with this enzyme?
Regards
Carlos
Not familiar with that particular product, but I recall having problems with no restriction when moving away from using NEB and Promega enzymes also. I found that it was better to spend the money on enzymes and try to save money elsewhere.
Since BcuI is an isoschizomer of SpeI, you could be seeing a problem because of methylated or unmethylated restriction sites in the Pseudomonas. Some isoschizomers will cut one site but not the other (methylated or unmethylated). For the record, I have not performed PFGE on Pseudomonas, but I did see what I believe were similar problems in Campylobcater.
Hope you find a solution soon.
Robert
Thank you for the answer, Robert.
After several attempts, 2uL worked well. And TBE + thiourea.
Regards
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