I have a plasmid with 2 AarI digest sites about 7bp apart to allow for the insertion of a fragment. The plasmid is 14000bp long and when linearised I presumed would be a single band larger than undigested plasmid open conformation band? when I did digest as to the protocol in http://www.nature.com/nprot/journal/v9/n10/full/nprot.2014.157.html (pretty much the same as the manual) for 16 hours I had 5 bands (as attached) and the top 2 where identical to undigested bands.
So is the top band in the digest the right one or do I need to opitimise it?
Thanks
Hi there,
If the upper band of the 1kb ladder is 10kb then the upper band in the digest is the linearized plasmid. To be sure of this, you should run gel for longer and using ladder going beyond 10kb (at least up to 15kb). Your issues are : the digest is not completed as supercoiled DNA persists (lower band of the intact plasmid), it might be because of close proximity of AarI sites and you may also be confronted to AarI star activity (appearance of unwanted bands). So I would suggest to repeat the digest for the same time but with less enzyme (make sure you use the right plasmid/ enzyme ratio to avoid the star activity).
Thanks Dominique,
It is indeed a 1kb ladder. I digested the enzyme with 3 other enzymes which all cut at one site (see image attached, the digest bands were all aligned but went a bit wobbly in gel) from the gel, looks like the top band is the right one. I couldnt find any info on what the ratio of enzyme to plasmid should be but ill decrease enzyme and keep time the same.
Depending on the company you bought your enzyme, you might look into methylation. AarI as supplied by thermo is sensitive to CpG methylation. That will prevent any digestion unless you've taken the right steps. I've found it easiest to design primers to add a different site, but if that's not an option alternative is to make sure you are isolating your plasmid from a strain mutated to not methylate DNA. The downside is these strains can be more prone to mutations.
As for DNA/enzyme ratio, I believe as long as there is no star activity more enzyme and longer incubation can't hurt. Compare the "unit digestion" for the enzyme you purchased with incubation time. Usually the enzymes are so efficient that even 0.5 uL is an overdigestion at 1 hour, but it doesn't usually matter. The product sheet usually lists all this, but just make sure your above the unit digestion and there's no star activity and the DNA/enzyme concentrations shouldn't matter. NEBiolabs has lots of good info for all their enzymes, some are better for longer digestions and others aren't; https://www.neb.com/tools-and-resources/usage-guidelines/restriction-endonucleases-survival-in-a-reaction (they don't have AarI, but wherever you got your enzyme from probably has something similar. Brand names matter!)
Hi there again,
Normally you should be able to get the enzyme user guide from the company selling the enzyme... The only company I know selling AarI is ThermoFisher and indeed as Bryce mentioned earlier it's sensitive to CpG methylation and exhibit star activity if more than 1U is used per µg of DNA...
Something strange here, I think. Were all 6 lanes of the digested plasmid in the first pic the same?? And in the second pic, did you digest the same amount of plasmid with all 3 RE?
What I don't understand is why you appear to have both incomplete digest and star activity. Is the plasmid commercial or lab-prepared?
Even if you get the digest to work, how will you prevent self-relegation and also determine the orientation of the insert? Are there other sites in the plasmid that you might be able to exploit?
I agree with the above, specially with Geoff's remark. If there is any chance, try to find a strategy without this lousy enzyme. To get a vector, the enzyme has to cut twice. With an enzyme that cuts 8 nucleotides 3' from the recognition sequence, it makes me worry that your cuts may interfere.
The only good thing is that you don't have to worry about CG methylation when handling a plasmid.
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