In my last article, I explained that plasmid DNA recovered from a plasmid prep consists of few different species; supercoiled, nicked, linear and single stranded circular, and how you can distinguish them on a gel.
Supercoiled DNA is the desired form of plasmid DNA; it performs better in downstream applications such as automated sequencing and transfection and also transforms more readily. Commercially, nicked and linear forms of plasmid DNA are considered impurities in large-scale preparations for DNA vaccines or plasmid DNA therapies.
Although supercoiled is normally the predominant species, nicked and linear DNA forms are usually recovered as minor species in all plasmid preps. However if you don’t do things quite right they can become a significant portion of some preps. Which is not good. Not good at all. And if the prep goes spectacularly wrong then you can end up with a prep full of the single-stranded stuff, which no-one of us want to see – do we?
So here I am going to give you some tips on giving your plasmid preps the TLC they deserve so you can minimize the proportion of the non-desirable species, and maximize your recovery of the lovely super-coiled stuff.
1. Harvest your plasmid prep bacterial culture in the stationary phase
Nicked DNA is often seen when bacteria are harvested prematurely during logarithmic growth. To avoid isolating large amounts of nicked DNA, harvest bacteria that have become saturated and have entered into stationary phase (after 12-16 hours of growth).
2. Vortexing and pipetting your plasmid — take it easy!
Nicked or linear DNA may occur due to mechanical shearing of DNA if preps are vortexed or shaken too vigorously during isolation of the plasmid. So take it very easy; mix gently, don’t vortex and pipette softly and sparingly.
3. Never over-cook the alkaline lysis
As you will know by now (or if you don’t check out this) alkaline lysis is your friend for isolating plasmid DNA from genomic DNA but if you over-do it you can permanently destroy your plasmid by permanently denaturing it in the single-stranded circular form. But never fear, just keep the incubation time for the lysis to the recommended length and be very gentle with the mixing at this step, and you will be fine.
4. Handle plasmid re-suspension with care
Shearing may occur during resuspension of the plasmid after air-drying. The larger the plasmid, the more likelihood that shearing can occur. Be gentle with the preps after precipitation and allow DNA to absorb into water rather then pipetting.
5. Avoid the endonucleases
Alternatively, certain strains of bacteria contain endonuclease A (endA+) that can nick or linearize DNA. If the plasmid cannot be moved to an endA– strain, then perform the plasmid prep as efficiently as possible to move the DNA away from the enzyme quickly.
6. Stay cool and get more supercoiled plasmid in your prep
Finally, it has also been shown that performing all steps at lower temperatures (4-12°C) increases the amount of supercoiled plasmid recovered (Carbone, et. al., 2012. Biochimica Polonica. 59: 275-8.)
Improving your technique and paying careful attention to small steps can increase your supercoiled DNA recovery and give you better results down the line. All this in return for a little TLC – it’s not too much to ask, is it?