Hi there,

I've no experience with this pair of RE. How close are the two sites? There might be a competition between proteins for DNA resulting in digest inhibition.To have a clearer view, you should run individual digest on your favorite DNA which I guess is circular plasmid and in parallel on known substrate (to check RE activity) and see what happen : it will tell you if there is competition issue (enzymes should work OK individually) or a plasmid structure issue (one or both enzymes don't work on your fav DNA whereas they perfectly work on model DNA).

Hi there,

I've no experience with this pair of RE. How close are the two sites? There might be a competition between proteins for DNA resulting in digest inhibition.To have a clearer view, you should run individual digest on your favorite DNA which I guess is circular plasmid and in parallel on known substrate (to check RE activity) and see what happen : it will tell you if there is competition issue (enzymes should work OK individually) or a plasmid structure issue (one or both enzymes don't work on your fav DNA whereas they perfectly work on model DNA).

Acc65I is dcm and CpG methylation sensitive. Could this be a problem or are you cutting pcr product. I assume you are cutting in NEB 3.1 buffer or equivalent which seems to be the only buffet giving 100% activity for both enzymes. Dominique,s idea is very good.

Thanks for these answers to both of you. @Dominique, that's a good idea about steric inhibition, there are just ~60 bp spacings between my restriction sites; I will set up sequential digestions in parallel to see whether that makes a difference. @Paul, I am cutting cloned plasmids, but think that in my sequence there should be no dcm-mediated methylation (as far as I know, Acc65I recognition sites are only methylation-sensitive in combination with particular surrounding sequences, but please correct me if I am wrong, as that would of course make a big difference.)

Hi Patrick,

I think the methylation idea only works id there is a C in front of or a G at the end of the ggTAcc  recognition/cutting site and methylation occurs . If the sequence is other than that or the plasmid is in a dcm negative line then there should be no problem

1. How could you be sure that the double-digestion did not work? Separated by only 60-bp between these two restriction sites, sometimes, it will be hard to see the obvious difference of movement between the 'cut' and 'uncut' plasmids on a gel. Due to the small size, the 60-bp band might also be hard to visualize at the bottom of a gel.

2. Why the digestion time was so long, 15 hours overnight at 37oC? Prolonged reaction time can cause 'star activity'. [https://www.neb.com/tools-and-resources/usage-guidelines/star-activity].

I think that the 60 bp excised band will be very much weaker than the plasmid fluorescent band(s) because the longer plasmid will trap much more ethidium bromide but it should be visible if it is there. Often with uncut plasmids they adopt many stable shapes and run at 2 or more different apparent sizes. The tension is relaxed on single cutting of the plasmid to form a single sized fragment so there may be a change from multiple bands to a single band if even one of the enzymes cuts the plasmid

With 'single' or 'double' cut (60-bp out), the resulting band should run a bit behind the band of the uncut (see the 2 attachments). Picture 1 shows results with 'single' or 'double' cuts, while Picture 2 shows 'single' BamHI cut. Have you seen the similar  phenomenon in your gel? Or the 'cut' band looked just like the 'uncut' band?

Can we take a look at your gel?

Hi again,

Linearized plasmid always runs slow compared to circular supercoiled plasmid and runs always faster than circular relaxed plasmid (supercoiled and circular relaxed forms being the major plasmid structures). So whatever the situation, a plasmid linearized by a single cleavage can be spotted unambiguously.

Thanks for all your suggestions. Just to clarify: My vector has in fact 2 Acc65I and 2 NotI restriction sites, between which there are ~60 bp; however, these don't matter to me: it is the two longer of the 4 resulting products (~3.4 and ~2.8 kb) that I need to separate. The gel showed two bands at roughly corresponding positions, but they did not separate well enough to cut them. @Dominique: I set up sequential digestions and the gel looked the same as for double digest. @ Yuan-Yeu Yau: Sorry, I did not take a picture of the gel, will do next time if the problem persists. I use over night digestion