I want to digest pET28a plasmid (5369 bp) with 2 restriction enzymes (RE)- EcoRI and XhoI for cloning. I checked the REs have single cutting site of each in the plasmid. However when I digest it with EcoRI for 2 hours, it gives 2 bands- 5369 bp (desired) and ~5000 bp (lane 1).
If the construct has an insert that created another EcoRI site then it is now ~10kb plasmid. Interestingly, XhoI digests the plasmid completely (loaded full reaction volume) and gives one sharp band of desired size (5369 bp) not 10kbp (lane 2).
And again in case of double digestion, the undesired band appears again (lane 3).
If it is supercoiled plasmid, then why does it appear in case of EcoRI only? I changed EcoRI brands, incubation time, buffers..but same result.
pET28a: 14ug
EcoRI (invitrogen): 20U
XhoI (NEB):20U
10x Cutsmart buffer (NEB): 2 ul
Rest volume: NF Water
I am just guessing here but I wonder if you are seeing some EcoRI* activity where the enzyme is cutting at a second site that is not really a recognition site but is partially recognized. Since digestion at the second site is very inefficient you get some plasmid cut one time and a fraction cut twice.
EcoRI* activity is sometimes triggered if there is too much glycerol and too much enzyme. For example if you are adding 1ul of enzyme to a 20ul reaction then you may be at the glycerol limit. And with the double digest you have even more glycerol.
Try using less enzyme and see if the problem goes away, you should only need 1-2 units so add just a fraction of the enzyme (or increase the reaction volume).
Could be a partial digest so you are seeing circular and linear bands.
I would advise run the uncut plasmid in parralel to see the size and pattern of the plasmid. I agree with Robert that it seems like the EcoRI is not cutting properly, hence two bands (one from uncut supercoiled). Also, EcoRI has only half activity in Cutsmart buffer, only EcoRI-HF works 100% in cutsmart. For normal EcoRI you need buffer 2.1
Furthermore, 14ug is too much DNA, and although company suggest 1U cuts a 1ug in one hour, i would suggest 10U for 1ug as freeze thaw cycles affects this. Decrese the amount of DNA (1-2 ug is more than enough for digestion analysis) and incresae either enzyme conc. or time (might show star activity as these are not HF version of enzyme).
You could gel-excise and purify the band that is the size you want and call it done. You'll sequence the product again anyway to be certain it's what you want.
Words of wisdom: sequence the plasmid for yourself. Never trust a plasmid that you got from ANYONE else. Trust me.
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