Most of the time, plasmid prepping is a breeze. You get your stab from Addgene, streak for single colonies, sub-culture, and prep with one of the many commercially available DNA prep kits or your lab's favorite in-house protocol. DNA yields for this procedure are typically in excess of 100 ng/ul, more than enough DNA to proceed with most applications, such as PCR, cloning, transfection, or long-term storage. But what about those pesky situations where your plasmid yield is sub-optimal? If you have already purifed your plasmid, you can try to concentrate the DNA using a speed-vac, ethanol precipitation, or other chromatographic methods. But wouldn't it be nice to avoid an extra concentration step? If you are consistently getting sub-optimal plasmid yields from your prep, you may want to consider optimizing your growth conditions. In this blog, we will outline many of the variables that could affect DNA yields and suggest steps to super-charge your plasmid preps.
The copy number of a given plasmid refers to the number of copies supported within each bacterial cell of your culture. Copy number is ultimately limited by the plasmid’s origin of replication, but will also be affected, not only by the size and nature of the insert, but also the propagation strain, growth conditions, and inoculum (see below). To further complicate matters, plasmid copy numbers are often ambiguous at best and many times should be viewed more as a guideline than a rule since they are typically based on the copy number of the empty plasmid backbone or a researcher’s personal observations. If you are experiencing low yields from a plasmid whose copy number is unknown, it is entirely possible that you are working with a low copy plasmid.
When working with low copy plasmids, the first step to improving yield is often to start with more culture volume and many common plasmid prep kits explicitly state this in their protocols. A higher volume of lysate can be passed over the binding matrix, improving yield in the eluate; however, you do need to be cautious to stay within the recommended range of the filter. Overloading the column with too much lysate could clog the matrix and inadvertantly lower the amount of DNA you purify! If you've reached your lysate volume limits, the copy number of some some origins of replication (e.g. p15A, ColE1), can be increased by adding chloramphenicol to the culture medium.
There are many commercially available strains for the propagation and purification of plasmid DNA, and often it is difficult to know which will work best in your particular experiment. For example, certain E. coli strains such as HB101 and its derivatives may be appropriate for long term storage, but these also produce carbohydrates, which could reduce lysis efficacy and plasmid yield. Other strains, such as Stbl3, are engineered to reduce recombination and therefore better propage unstable plasmids, but are also endA+. endA encodes a thermostable periplasmic endonuclease. If not properly removed during the wash steps of your purification protocol, EndA can co-purify with your plasmid DNA, and may shear the plasmid causing a smear when run on a gel.
DH5alpha has consistently been shown to give good, high quality preps due to its endA1, recA1, relA1 genotype, as these mutations improve plasmid stablilty and yield, and this strain (or one of its derivatives) is recommended whenever possible. While choice of strain will ultimately depend on the particular features of your plasmid, it is advisable to check out the genotype of your E. coli to ensure it is suitable for your application. This publication looked at the DNA quality of plasmids isolated from different E. coli hosts. Addtionally, we have previously reviewed many common lab E. coli strains on the blog, and also recommend this handy guide from OpenWetWare for a more extensive lising of common E. coli strains.
Culture Media, Antibiotics, and Inoculum
Culture conditions play a critical role in plasmid DNA yield as is demonstrated in the figure on the right. Different plasmids and strains will vary in their optimal growth conditions. For many high copy plasmids, standard LB broth and antibiotic concentrations will work just fine; however, for low copy plasmids or slower growing strains, a change in media may be advantageous. Low copy plasmids produce fewer transcripts of their antibiotic resistance genes, and can therefore be cultured in media containing half the normally recommended antibiotic concentration.
Additionally, you can achieve higher cell density using a nutrient-rich broth such as Terrific Broth, 2xYT, or a home-brew optimized for plasmid yields (e.g. H15). Spiking your media with supplements such as magnesium salts, buffering agents, and/or supplying additional carbon sources such as glycerol or glucose (in moderation) may also serve to increase cell density and plasmid yield. For best results and highest DNA yields, it is always recommended to start from a single, fresh colony. Sub-culturing directly from a frozen glycerol stock or agar stab may lead to loss of the plasmid, and using older plates could increase plasmid loss or mutation.
Time, Temperature, and Oxygen
Lastly, you can optimize your growth time, temperature, and shaking speed to maximize cell density and plasmid yield as these factors are generally, but not perfectly, correlated. Typical growth times for high copy plasmids in standard growth strains range from 12-16 hours, but cultures with lower copy plasmids often need to be grown for 20 hours or more to achieve maximum plasmid yield. The optimal growth time should be determined for each plasmid/strain combination individually, either by measuring OD or, ideally, by harvesting/miniprepping at various timepoints.
Additionally, certain plasmids and strains are best suited for growth at temperatures other than the standard 37℃. This should be indicated in the plasmid information or growth strain instructions from the manufacturer. Finally, you will want to make sure your culture is getting the right amount of oxygen for optimal growth as insufficient gas exchange will prevent your cultures from reaching the desired density. The volume of your flask or culture tube should be at least 4x greater than your total culture volume and you should ensure the shaker speed is fast enough for sufficient gas exchange in the overnight culture. Generally, large cultures in flasks may be shaken around 220 RPM, but smaller cultures, especially those in deep-well plates, require a faster shaking speed for proper aeration (260-300 RPM).
Additional Resources on the Addgene Blog
- Learn Various Cloning Techniques
- Get Tips on Analyzing Sequencing Results
- Learn How to Varify Your Plasmid
Additional Resources on Addgene.org
- Find Info on How to Quantify Your DNA
- Learn to Do a Bacterial Transformation
- Learn How to Perform a Diagnostic Restriction Digest