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How to Maximize the Quantity and Quality of Purified Plasmids

photo says "Maximum" - DNA extraction article

Before spin columns, PCR, and reaction miniaturization, being able to maximize plasmid prep DNA yield was a highly competitive lab accomplishment. A card-carrying Molecular Biologist had to be able to routinely get 500 mgs of DNA from a 1 L culture (1 g really showed your prowess). These days, many applications can and should be accomplished with a DNA extraction protocol that yields <1 ng of plasmid DNA. Occasionally, however, there are still times when large, clean plasmid batches are required. For example, one great DNA extraction prep can make or break a good transient transfection cell assay or a library construction. Assuming you are working with a mid- to high- copy plasmid, getting 1 g of DNA should be easy. Here follow a few tips from “back in the day” that today’s Molecular Biologists might not be aware of.

Start Fresh

First and most importantly—start with a fresh transformant. Start from DNA (mini-prep is fine) and re-transform competent cells. Do this even if you have a frozen glycerol stock or old colony on a plate. Grow only one night and plate at different densities so in the morning you have discrete, single colonies. Pick a healthy colony from that overnight plate (do not refrigerate) and inoculate your 1 L culture. While anecdotal, we tested stocks versus fresh transformant in a side-by-side comparison one summer in our lab. The fresh transformant won hands down. Using a fresh transformant to start your bacterial culture can double your plasmid yield, so it is worth taking the time for this extra step when you are talking about 1 g vs. 0.5 g.

Aerate Your Culture

Second—shake well. Make sure that the incubator shakes enough to raise a nice foam on that culture and you are growing at no more than 37°C (test with a thermometer). No slow, lazy shakers or rotators.

I have heard legend and lore that growing bacterial cultures at a lower temperature can be beneficial, but I have not seen this help in relation to plasmid yield. However, it can make a big difference when making your own competent cells, though.

One other tip: pre-warm the media before inoculating so you will have a consistent procedure each time.

Grow Until Saturation

Third—don’t over grow the culture. This seems to be the key to keeping the quality of the final prep as high as possible. Harvest the culture when it has just reached saturation, before cells start dying. If you inoculate with a single colony late in the evening, you can sometimes 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 every 30 minutes and stop the culture when the readings stop changing drastically. Saturated cultures can go as high as OD600 = 18 if you let them, but this is always accompanied by cell death. You really want to harvest closer to OD600 = 1 and/or before the culture reaches a plateau for more than 30 minutes. Don’t be greedy—it will backfire.

Don’t Stop

Fourth—don’t freeze the pellet. For most purposes, you can freeze bacterial pellets and complete the plasmid prep later without a problem. To maximize yield, however, don’t stop—keep going until you have purified the DNA or at least have it precipitating in ethanol. This also seems to help with quality of DNA when you use a spin column, too.

Know Your Preparation Method

Whichever DNA extraction method you use, learn the chemistry behind it. Learning how the method works will help you with troubleshooting later. For example, in the alkaline lysis method, if you vortex or mix the sample too vigorously, then you’ll shear the genomic DNA. Why is this a problem? Separation of plasmid from genomic DNA in this DNA extraction method relies upon the genomic DNA being too large to re-anneal. If you have short stretches of genomic DNA, then those can re-anneal. This will contaminate your plasmid prep with genomic DNA.

One Final Tip to Maximize Your Plasmid Yield

One last little tip for the newbies: don’t accidentally toss your plasmid DNA. If the last step of your plasmid prep protocol says to “rinse” the pellet with 70% Ethanol, then this actually means to add 70% ethanol, spin hard in the centrifuge, and carefully remove the supernatant by gentle aspiration before drying. This does not mean flicking the 70% ethanol into the sink like when you rinse a glass. One technician in our lab was getting abysmal plasmid yields for weeks. It turns out he was flicking his grams of plasmid down the drain.

Do you have any other tips for maximizing your plasmid yield? If so, let us know in the comments!

1 Comment

  1. NALLAMOTU KRISHNA CHAITANYA on February 5, 2020 at 11:10 am

    Wow, Let me add a couple of more, in our lab, we warm nuclease-free water at 42-50 degrees before adding it to spin column for eluting plasmid, which is believed to increase the yield. One more tip, after elution we pipette it back and add it to the column once again if any plasmid stuck to the column it will elute out.

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