Why did I get different restriction digestion results from three plasmid preparations, even though they all came from the same single colony of BL21(DE3) carrying the pET-SUMO-Lcp3 plasmid?
I cultured a single colony in LB + kanamycin, then inoculated three separate LB + kanamycin liquid cultures from it. After plasmid extraction, I digested the three plasmids with NheI and HindIII. Only pET-SUMO-Lcp3 NO. 1 showed a clean and correct digestion result. In contrast, pET-SUMO-Lcp3 NO. 3 also showed both vector and insert bands, but there was an extra band above the vector and a faint smeared band between the vector and insert (as indicated by a red arrow).
Since they all originated from the same single colony of BL21(DE3), what could have caused this difference?
# I’m certain that NheI and HindIII each have only one restriction site on the pET-SUMO-Lcp3 plasmid.
Seems like a problem with the reaction mix assembly. Plasmid in lane 2 is barely digested, the supercoiled and circular relaxed plasmid forms are still there. Smear in lane 3 might be because of overload with one of the enzyme (star activity or so). But the plasmid is likely the same, just digested to different extent. Schematically:
lane 1
enzyme1 concentration = OK
enzyme2 concentration = OK
lane 2
enzyme1 concentration = low
enzyme2 concentration = very low
lane 3
enzyme1 concentration = low
enzyme2 concentration = high
Victor G Stepanov Thank you for your response. So you mean that all three plasmid samples were digested and showed the expected bands, but lane 2 wasn't fully digested, and lane 3 showed signs of star activity. However, what confuses me is that I used exactly the same digestion mix for all three lanes: 10 µL plasmid (35 ng/µL), 1.5 µL buffer B, 1 µL NheI (10 U/µL), 1 µL HindIII (10 U/µL), and 1.5 µL ddH₂O, for a total volume of 15 µL. All reactions were incubated at 37 °C in an incubator for 2 hours and 30 minutes. The enzyme amount should be sufficient. Additionally, I later found out that the band larger than 10 kb might be caused by genomic DNA contamination—could that be the reason?
When you pipet small volumes of viscous solutions, you must pay special attention if the solution entered the pipet tip at all, and also make sure you didn't get too much of it on the outside of the tip. Just don't immerse the tip too deep into the enzyme stock and check it by eye after withdrawing. It's easy to mess up with the enzymes because they usually supplied in a glycerol-containing buffer, which is viscous enough to make precise pipetting troublesome.
If you want to repeat the experiment, I would recommend to pre-assemble all of the components (other than the plasmid) in a stock, and start the digestion by mixing aliquots from this stock with plasmid solutions. This way you can avoid pipetting small volumes, and the final enzyme concentrations will be all the same in all three reaction mixtures. In your particular case, you can make such a stock by mixing 4.5 mcl H2O, 4.5 mcl buffer B, 3 mcl NheI and 3 mcl HindIII together in the order as written. This would yield 15 mcl total. Mix the stock well by pipetting, and add 5 mcl aliquots from it to 10 mcl of each plasmid prep. If you want to play safe, it's better to slightly increase the amount of the prepared stock to leave some room for pipetting errors. I would make 4.8 mcl H2O, 4.8 mcl buffer B, 3.2 mcl NheI and 3.2 mcl HindIII to end up with 16 mcl of the stock, which would give you 3 aliquots x 5 mcl for the restriction digests + 1 mcl leftover as a reserve.
Genomic DNA doesn't enter agarose gels that deep, not to say it's not supposed to be in your plasmid preps if plasmid purification was performed properly. You may get some smear of large gDNA fragments if you vortexed solutions during purification procedure following the cell lysis step. But they are not seen as a sharp band. Some protocols may be prone to leave some gDNA as a contaminant in plasmid preps but this gDNA is usually linear or relaxed circular due to shear during purification procedure. These forms would just remain in the well 'cause they are too long to enter the gel. The only gDNA form which can realistically travel through agarose is the intact tightly-packed nucleoid DNA. It's pretty unlikely to get it unintentionally in sufficient quantities. And even if you got somehow gDNA in your plasmid preps, it's going to be digested by restriction enzymes into a ladder through the entire lane.
Victor G Stepanov I see, thank you for the suggestion. I will prepare the restriction digestion master mix as you described and try the digestion again to see if it improves the results.
Hi Lorenzo,
I think the recommendations from Victor G Stepanov are quite reasonable. However, I would consider one additional point regarding your problem. From your description it seems that you have done the same restriction mixture for all your plasmid preps (for three different liquid cultures), but with three different outcomes (and I agree with Victor that this looks very much like incomplete digestion). I don't know how you did your plasmid preparation, however the quality of your preparation could also influence the results of your digestion as contaminations might inhibit your restriction enzymes. This might be an alternative explanation why you get 3 different results with three different plasmid preparations. So if you still have problems after following the recommendations of Victor G Stepanov I would consider doing new plasmid preps (or only using the first one for subsequent applications).
Thank you all for your suggestions.
The problem has been resolved after repeating the experiment.
Much appreciated!
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