After the initial excitement of growing and isolating plasmid DNA, routinely preparing plasmid mini/midi/maxi preps gets boring. You definitely want a way to squeeze the maximum amount of plasmid DNA out of your culture. Good news—you can increase your plasmid yield using antibiotics.
Keep the Pressure On to Improve Plasmid Yield
Remember that you need to maintain selective pressure in the culture for your plasmid at all times. Otherwise, you will start losing the plasmid from the culture. So, include your antibiotic or other selective agent in the medium when growing your culture.
One special case is if you’re selecting for the plasmid with ampicillin. The plasmid produces the ampicillin-destroying enzyme, beta-lactamase, that accumulates in the medium during growth. To remove the beta-lactamase, get rid of the starter culture medium by pelleting the cells and re-suspend them in fresh medium before scaling the culture.
Many commonly used vectors have a relaxed origin of replication, which allows for decoupling general protein synthesis from plasmid replication in E.coli. Adding chloramphenicol stops protein synthesis, but the plasmid will continue replicating. This will result in many more copies of your vector per bacterial genome.
The relaxed origin plasmids that I am talking about have the pMB1 or ColE1 origin of replication. The most common vector backbones with this origin are (in descending order of copies per cell):
pACYC – make sure that your derivative is not chloramphenicol-resistant, as this will make adding the antibiotic useless in any concentration.
There are two ways of using chloramphenicol for your plasmid amplification.
1. Use Chloramphenicol According to “The Maniatis”
This is a recipe from the classical protocol cookbook by Maniatis et al. aka “The Maniatis.”1
Maniatis recommends growing your culture until saturation, then add 170 µg/ml of chloramphenicol, and continue growing the culture for a further 16 hours. You will stop protein synthesis completely in an already dense culture. The cells will stop growing, but the vector will keep amplifying.
2. Use Chloramphenicol According to Begbie
(No, you’re not supposed to know Begbie as it’s not yet a household name).
Begbie et al. have explored another, faster, possibility.2 In the previous protocol, growing the culture will take you at least 36 hours.
The alternative is adding a much lower concentration of chloramphenicol, 3 µg/ml, when you inoculate the main culture with your starter one. The sub-inhibitory concentration will slightly slow down your E.coli doubling time, but will not stop it. However, it will increase the copy number of your vector several times.
Irrespective of the way you amplify your vector using chloramphenicol, treat the resulting culture as containing a high copy number vector. First of all, don’t overload the midiprep or maxiprep column with lysate—use the minimum volume of culture according the protocol. Elute with maximum volume of buffer and repeat the elution. You can always concentrate DNA later.
Do you have other tips and tricks using antibiotics to improve plasmid yield? Please, share it with the community in the comments below.
Maniatis, T., Fritsch, E. F. and Sambrook, J. Molecular cloning.New York: Cold Spring Harbor Laboratory; 1982. pp 545. [back]
Flow cytometry is a fluorescence-based technology, as is fluorescence microscopy and confocal microscopy. Fluorescence is fundamental to how a cytometer gathers data, but I am often surprised, as a core manager, at how little new users know about the process of fluorescence. So, this is where I always start the training process. Let’s get physical […]
It’s great to have you in the Bitesize Bio family! We’ve sent you an email to confirm your registration. Please click on the link in the email or paste it into your browser to finalize your registration.
For more information on how to use Bitesize Bio, take a look at the following image (click it, for a larger version)
An error occured while registering you, please reload the page and try again