I am trying to insert my fragment in pET21 plasmid, but if I do not reverse pET21 plasmid in CLC or other software, the direction of restriction sites will be disrupted. I was wondering if anyone could explain me the reason.
Thanks a lot.
Dear Nasrin,
Usually restriction sites are palindromic sequences so they should not be disrupted. There might be a problem with a plasmid sequence (polarity of DNA strands?). Please describe your problem in details.
Best wishes
Genbank record of pET21 has lacI sequence on plus (+) strand but MultipleCloningSites and amp resistance gene are on minus strand (-).
If you are using software to check your experiment by in silico cloning you should not have to reverse your pET21 sequence.
You can reverse (complement) your plasmid sequence at the beginning or at the end of your procedure for better visualisation but for cloning program it does not matter.
While not necessary, it is more natural to work with a plasmid sequence in the orientation where the DNA elements are ordered: promoter...cDNA...terminator. You can easily reverse the whole sequence or just the insert with WebDSV, an online DNA sequence editor:
http://www.molbiotools.com/WebDSV/
- Badges
- Science topic