I have a vector with PfoI site. I did single enzyme digestion to check for the linearity. For the reaction, I used 5U of thermo scientific PfoI for 1ug of plasmid with an incubation time of 14hours. After the reaction was complete, I loaded entire sample on an agarose gel and was surprised to see two bands. Can anybody explain what could be reason behind this extra band. I feel it could because of partial digestion but I am not sure. I am attaching gel picture. Details are: first lane is control(undigested plasmid), [2,3,4,5] lanes are PfoI digested samples, and remaining lanes can be ignored.
PfoI cleavage is impaired by overlapping dam methylation. (and blocked by dcm methylation)
Did you check whether your site is methylated?
Then indeed, on the first sight it looks like the digest is not complete… Hmm the only thing that I can think of at the moment is either to increase the amount of units used for the digest, or re-check that the buffer system you are using gives 100% activity. But guess this you have already done.
I agree that it's incomplete digestion so use more units or incubate the reaction longer, adding PEG will help keep the enzyme and plasmid in closer proximity, if that will not hinder your next step. I would do an additional 2 site digestion if you are checking the identity of the plasmid so that the linear and supercoiled bands can be better differentiated.
with fast digest enzyme from theromfiosher you can have better results. Try FastDigest (# FD1754).
I had written a much longer answer which addressed many interpretations of the data.
But there is one interpretation that should be addressed first:
PfoI cleavage is blocked by both Eco dam and Eco dcm methylation. Your message did not identify the E coli strain in which the plasmid was propagated.
The vast majority of E coil strains are dam+ and dcm+. Therefore, the vast majority of E coli strains will block cleavage by PfoI. One strain which permits digestion by PfoI is GM2163 (available from NEB). Or you can employ a similar strain that is deficient in both Eco dam and Eco dcm (i.e., it should be both dcm- and dam-). Most other E coli strains will block cleavage by PfoI.
Note: For PfoI sites, the occurrence of overlapping E coli dam methylation sites depend upon the sequence of your plasmid. That overlap can occur within either of two orientations. In particular, if your PfoI site is preceded by a GA or if your PfoI site is followed by a TC, then Eco dam methylation is relevant. So, being most precise, if your PfoI site is either GATCCNGGA or TCCNGGATC, then Eco dam methylation will block cleavage within your plasmid.
paul r walsh
P.S. I worked at NEB for 20 years where I often assisted customers regarding DNA methylation causing blockage of cleavage by restriction enzymes. But, if you want authoritative information about any particular restriction enzyme, then you should contact the manufacturer. If you do, then I encourage you to cite the information I offered.
Thank you for your suggestions. If we consider it to be partially digested, which one could be a completely digested band of the two bands. Would it be possible to tell this from the gel picture?
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