I am trying to digest a pET-11a SUMO vector with Kpn1 and BamH1 (expected weights of both bands approximately 5770 bp and 210 bp, respectively), but I am getting mixed information on the NEB online site for the enzymes and their incubation times. The other vectors I’m working with appear to work fine (PUC vectors) for 2-hour digests. I was digesting using 500 ng of plasmid and the concentration is 144 ng/ul.
The NEB site says that BamH1 has 75% activity in 1.1 where Kpn1 has 100, but also says if the buffers are not compatible to use more of one of the enzymes in the digest. It also says BamH1 is a time-saving enzyme but after running the gel for 2 hours the vector still is not completely digested.
if anyone has encountered this problem or has digested these kinds of vectors, which information should I go with?
Hi there,
If the double digest is working fine with pUC then there is something going wrong with the plasmid pET11a SUMO you use. According to a quick search there is no KpnI site neither in pET11a nor in the 3 pET11a-SUMO I could find from Addgene site...
https://www.addgene.org/search/advanced/?q=pET11a-SUMO
Thank you all for your feedback, it was very helpful.
I forgot to add something to this question. I forgot to add that the Kpn1 site is in the insert of the vector provided to me (attached).
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