Mystery band after vector digestion - (Oct/07/2005 )
Hi all. I am preparing 4 variations of BRET (bioluminescent resonance energy transfer) vector for ligation and I am digesting them with SacII and BamHI. The vectors are about 5kb each and when I digest with BamHI single digest for 2 hrs I get a single 5kb band. However, when I use the SacII for single digest control, I see one band at 5kb and another at 10kb. The predominant pand of the undigested control runs at about 3kb. I am really confused as to what this 10kb band is. Does anyone have any suggestions? Thanks.
Wow, this is a wierd one, huh? Perhaps you're seeing plasmid dimers that are cut only once with SacI? If you digest two equal aliquots, one with BamHI and the other with SacI, run the gel, stain and photograph, does the intensity of the 5 kb band plus the 10 kb band in the SacI lane equal that of the single fragment in the BamHI lane?
From NEB (emphasis added):
Sac I is inhibited by salt concentrations > 10 mM. Mini-prep DNA containing residual salt is resistant to cleavage. A 70% alcohol wash or dialysis can be used to remove the salt.
Sac I is sensitive to cytosine methylation at GAGmCTC but not GAGCTmC and insensitive to adenine methylation at GmAGCTC.
So perhaps one site of the dimer is methylated at GAGmCTC, and the other is not?
I ran into a similar problem with an enzyme once (Asp718, perhaps? -- it was a few years ago) where some sites were cutting and others not (from genomic DNA). Man, I puzzled over that Southern for a while. As I recall, I overcame it with an isoschizimer (KpnI?) that was not methylation sensitive...
SacII should not be methylase sensitive in e coli. Regardlesss this is a weird one. I have to think that perhap you have not cut to completion and are seeing nicked DNA?
Oh -- I misread the enzyme before. I thought you were using SacI -- tap14 is right, methylation shouldn't be a problem with SacII in a bacterial background. Interestingly, New England Biolabs has this to say about SacII:
Sac II needs to interact with two copies of its recognition sequence to cleave. As a result, substrates with single Sac II sites are cleaved at a reduced rate.