I am trying to digest a plasmid having 1.5 kb insert with NdeI and XhoI but I see only a single band at large size. Plasmid was expected to having these restriction sites. So what can be the reason for no digestion of plasmid.
Hi there,
I would first suggest you to check the activity of the 2 RE individually (on this plasmid and on a control plasmid bearing both sites). If they are both working OK in the same condition then it might be a sequence problem (mutated RE site). In order to check this, check the sequence. If everything is OK then it might be a topological problem (accessibility of the cutting site for one of the enzymes) but as you manage to linearize the plasmid this problem should be overcome...
Hi
this link can help you
https://www.addgene.org/protocols/restriction-digest
yes i agree with above both comments along with that just suggest try to toy with time and buffer used for restriction digestion see if still results are negative.. if yes..then its case of mutation
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