I am trying to linearize one circular plasmid with NEB NruI restriction enzyme. I have sequenced my plasmid and confirmed the NruI restriction site. I am using NEB buffer 3.1 in the NruI digestion reaction. Same plasmid is easily getting linearized at alternative restriction site i.e. PvuI. What may be probable reasons not to digest my plasmid with NruI?
Hi there,
NruI activity is sensitive to dam methylation. Is the recipient strain used for plasmid prep dam defective? If not, plasmid is methylated and NruI site becomes unsensitive to NruI digest. If the strain is actually dam defective the situation is a bit more tricky : it might be an issue with the enzyme (have you got an other plasmid with NruI site for activity control?) or with the plasmid structure which might prevent NruI site accessibility (try to linearize the plasmid with PvuI which is working OK and then digest with NruI, if native plasmid structure is the problem then NruI should be fully active on the linear form)
Hi there,
NruI activity is sensitive to dam methylation. Is the recipient strain used for plasmid prep dam defective? If not, plasmid is methylated and NruI site becomes unsensitive to NruI digest. If the strain is actually dam defective the situation is a bit more tricky : it might be an issue with the enzyme (have you got an other plasmid with NruI site for activity control?) or with the plasmid structure which might prevent NruI site accessibility (try to linearize the plasmid with PvuI which is working OK and then digest with NruI, if native plasmid structure is the problem then NruI should be fully active on the linear form)
Two controls would be nice:
- the same plasmid with different NruI enzyme (either try to find some in near lab, or try your supplier, that your digest doesn't work, so if they could send you sample from different batch
Hi Tomas,
ER less active on supercoiled than linear DNA substrate have been listed by NEB:
https://www.neb.com/tools-and-resources/selection-charts/cleavage-of-supercoiled-dna
But obviously NruI is not affected (at least with the plasmid used for the assay...).
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