Six Tips for Maximizing Plasmid Yield: Increase the Quantity of Purified Plasmids

Before spin columns, PCR, and reaction miniaturization, maximizing plasmid prep DNA yield was a highly competitive lab accomplishment. A card-carrying molecular biologist was expected to routinely pull milligram-scale yields from a large culture, and a particularly hefty prep really showed your prowess.

These days, many applications can and should be accomplished with a DNA extraction protocol that yields far less plasmid DNA. Occasionally, however, there are still times when large, clean plasmid batches are required. For example, a DNA extraction prep can make or break a transient transfection assay or a library construction.

What you can expect in terms of yield depends on several factors: plasmid copy number, host strain, insert size and toxicity, culture medium and aeration, antibiotic selection, and your purification method (including column capacity). Here are a few “back in the day” tips that today’s molecular biologists might not know.

Tip 1: Start Fresh

First and most importantly, start with a fresh, well-isolated colony. Streak out and pick a healthy single colony rather than inoculating straight from an old plate. Standard kit protocols also start by streaking from a single colony on a selective plate.[1]

If you have a reliable glycerol stock, streaking from that is usually fine. But if you’re unsure, re-transform competent cells with purified plasmid (a mini-prep is fine). Those include an old or questionable stock, a plasmid that may be unstable, possible contamination, or disappointing yields last time.

If you re-transform, grow only one night and plate at different densities, so you have discrete, single colonies in the morning. Pick a healthy colony from that overnight plate (do not refrigerate) and inoculate your culture.

While anecdotal, we tested stocks versus fresh transformants in a side-by-side comparison one summer in our lab. In our hands, the fresh transformant won hands down, giving substantially better yields. Your results will vary with the plasmid, strain, quality of your stocks, and culture conditions. Still, when a big prep really matters, it can be worth taking the time for this extra step.

Tip 2: Aerate Your Culture

Second, shake well. No slow, lazy shakers or rotators. In our experience, a culture shaking hard enough to raise a bit of foam was a good sign! How well a culture is aerated depends on shaker speed, orbit, flask size, and culture volume. As a rule of thumb, kit protocols recommend vigorous shaking (around 300 rpm) in a flask with at least four times the medium volume.[1, 2] Grow at no more than 37°C (check with a thermometer).

I have heard legend and lore that growing bacterial cultures at a lower temperature can help, but I haven’t seen it improve plasmid yield. It can make a big difference when making your own competent cells, though.

A good idea at this stage is to pre-warm the media before inoculating. I can’t promise it boosts yield, but it makes your procedure more consistent from prep to prep.

Tip 3: Don’t Overgrow the Culture

Not overgrowing your culture seems key to keeping final prep quality as high as possible. Cultures left too long start to die and lyse. An oversized cell pellet also overwhelms the lysis buffers, which reduces yield and purity.[1]

There is no single “correct” OD600 to aim for. Harvest according to the recommendations for your strain, medium, plasmid, and purification method. Overnight cultures are commonly harvested around late log/early stationary phase; QIAGEN, for example, suggests about 12–16 hours of growth.[2] OD600 readings at that point vary widely by strain, medium, flask conditions, and even the spectrophotometer you use. So treat this number as specific to your own setup.

If you inoculate with a single colony late in the evening, you can often catch this in the morning. We know scientists love to come in at weird hours, so you will have to find the optimal times for your bacterial strain and your sleep schedule. If you really want to get good at this, take OD600 readings over time for your own setup and note when the growth curve starts to level off. Don’t be greedy. Letting it sit for hours past that point will backfire!

Tip 4: Don’t Freeze

In our experience, it pays to avoid freezing the pellet. Freezing bacterial pellets and finishing the prep later is perfectly acceptable for many plasmid-prep workflows. But when chasing maximum yield and consistency, we found it best to keep going until the DNA was purified, or at least precipitated in ethanol. This also seemed to improve the quality of DNA from spin columns.

Tip 5: Know Your Preparation Method

Whichever DNA extraction method you use, learn the chemistry behind it. Understanding how the method works will help you troubleshoot later.

Take alkaline lysis, for example. The NaOH/SDS step denatures both plasmid and chromosomal DNA. When you add the neutralization buffer, covalently closed plasmid DNA can renature and stay in solution. Meanwhile, chromosomal DNA, proteins, SDS, and cell debris precipitate together and can be spun or filtered out.[3,4]

That is why gentle handling matters. If you vortex or mix the lysate too vigorously, you shear the chromosomal DNA into smaller fragments. These are more likely to stay in the soluble fraction and contaminate your plasmid prep with genomic DNA. Mix by gentle inversion instead, especially after adding the lysis and neutralization buffers.[3]

Tip 6: Don’t Flush Your Work Down the Drain (Literally)

One last little tip for the newbies: don’t accidentally toss your plasmid DNA. Your plasmid prep protocol may end by telling you to “rinse” the pellet with 70% ethanol. This means adding 70% ethanol, spinning hard in the centrifuge, and carefully removing the supernatant by gentle aspiration before drying.

It does not mean flicking the 70% ethanol into the sink like you would when rinsing a glass. One technician in our lab was getting abysmal plasmid yields for weeks. It turns out he was flicking his hard-won plasmid down the drain…

Good luck with your experiments!

References

1. QIAGEN. QIAGEN Plasmid Purification Handbook (expected yields, culture volumes, growth time, shaking, biomass vs. lysis buffer).
2. QIAGEN. Growth of bacterial cultures. Plasmid Resource Center.
3. QIAGEN. Lysis of bacterial cells for plasmid purification. Bench Guide.
4. Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979;7(6):1513–1523. doi:10.1093/nar/7.6.1513