problem with digestion

[mrudulamariam]

Hi
I am trying to clone a 1 kb pcr product within Xba1 and Xho1 sites of pET32a vector. I found that Xba is not cutting the plasmid. Plasmid was isolated from BL21DE3 strain. Can it be due to dammethylation? how long do res. enzymes stay intact when stored at -20?

[javaid]

hi
i think the problem is with enzyme, n the enzyme vial has its expiry date written on it, so will be stable upto that time in -20. u may try by changing the enzyme vial.... hope this helps

[mrudulamariam]

On the vial it is given last assayed on 03-2005. Can i take it as expiry date?

[Duke]

Originally Posted by mrudulamariam

Hi
I am trying to clone a 1 kb pcr product within Xba1 and Xho1 sites of pET32a vector. I found that Xba is not cutting the plasmid. Plasmid was isolated from BL21DE3 strain. Can it be due to dammethylation? how long do res. enzymes stay intact when stored at -20?

XbaI is Dam-methylation sensitive. Its activity is blocked by the overlapping Dam-methylation. As BL21(DE3) is not Dam-methylation deffective (and a very unusual strain for plasmid isolation by the way) it is likely that the Dam-methylation interferes with the XbaI cutting.

You may prepare a new plasmidprep from E.coli like JM110 which is Dam-negative.

Restriction enzymes have an exp. date (as mentioned before). If this would be the same as the "last assayed" date, it would be a bit strange, that companies first assay their enzymes, then mark them as expired and then sell them to you, wouldn´t it?

Usually they are stable for 1-2 years (this really depends on the enzyme, the source, the supplier, the concentration, etc.) at -20°C in their storage buffer.

As this storage buffer contains lots of glycerine (prevents the formation of crystals), do not use the enzyme in more than 10% of the final volume of a restriction digestion reaction. Glycerol also inhibits enzyme activity, which could also be a reason for XbaI not cutting your plasmid.