Pimp Your Plasmid Growth Medium
I often wonder why it is that molecular biology researchers stubbornly refuse to change 4o-year old methods that, while work, are not as good as newer, faster and cheaper methods out there.
I suppose rational scientists often have irrational superstitions.
One example of an old method that could be improved is the growth media used for plasmid preparation.
The majority of us, throughout our university careers, have used either SOC, LB or TB, for recombinant plasmid propagation, typically in E. coli. LB or Luria-Bertani broth has been in use for almost 60 years or thereabouts, while SOC has certainly been in use for 2 decades.
Pimp Your media and Save Some Money!
But by adding in a few more ingredients or being more economical on others (especially yeast extract and tryptone) you could get a higher plasmid yield, quicker and with less money.
To counter the naysayers, nobody wants to make very complex media with 15 ingredients requiring filter sterilisation, as this obviously defeats the object of economy of time and budget. Indeed, there are trade-offs between optimising for biomass, plasmid yield, quality, stability and cost with the difference between protein production and plasmid production, since plasmid production requires only cell growth, division, and plasmid stability.
The good news is that Michael Danquah and Gareth Forde from Monash University down-under have devised a stoichiometrically optimised medium for plasmid production. PDM, supposedly yields under the conditions they tested, twice the amount of plasmid in both volumetric and specific yields compared to TB, and LB is left in the dust. Better yet, because it uses less tryptone and yeast extract, the cost per mg of DNA is roughly one-quarter of the cost of LB.
The recipes for LB, TB, SOC and PDM are shown below. If you decide to break with tradition and give PDM a go, be sure to tell us how it goes.
Note: Autoclave glucose, KH2PO4 and Na2HPO4 separately – if you want to know why check out Doesnât Play Well with Others – The Chemistry of the Autoclave.
Originally Published 28 April 2008, Updated and Republished 19 December 2014.
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In my experience, studier media recipes are the best for protein expression and also plasmid yields. I suggest try them and check for yourself.
Not all media recopies give same yield of plasmid, they vary because of the titre of cells. In addition, be cautious of limitations with plasmid purification kits, they are optimized with LB as reference. Generally, use less media when changing from LB and try for plasmid extraction. As a rule, we use studier media at 2ml for plasmid extraction with comparable yields with 5 – 10 ml of LB..
In found Macherey Nagel plasmid extraction kits give high yields probably because of high binding capacity.
Tried the optimised “R” version of the PDM media with C/N ratio 2.8 and the yield was half of LB! This is consistent with what has been reported by a subset of users above using the non-optimised C/N ratio version of PDM. I tried it with Top-10 cells, shaking at 30 degrees and also with 37 degrees with same results. Ill give it a go again with DH5 cells to check if its any good or just baloney! FYI all reagents were from Sigma.
Hey I know it’s been a year since this post, but did you figure out the lower yields? Why? and were you able to fix it?
For my part I will report that I think the calculations for the C N ratio to accomplish the PDMR are to add 5.454g of NH4Cl (~1.4g N) for the 10g of glucose (~4g of carbon in there) = 2.8. I did calculate to more sig figs according to my balance.
Liam, (or anyone) did you try the PDMR to better/ worse success than the PDM?
What if I need a low-salt version of media (for zeocin antibiotic), what should I avoid in the medium?
Some questions about this:
Why can’t you mix everything and autoclave? The only thing I read on here was that it “doesn’t autoclave well.” What does this mean?
How does one make the stock solutions (10X) when it’s in gram amounts? Convert to molar?
Does the pH need to be altered in order for the Na2HP04 to dissolve?
Sorry I’m a bit lost on how to go about making this đ
I see this question came in a while back, so it may be moot now, but if you are in a quandry as to how to make this, why don’t you just order it from Teknova? They have catalog # P0820-06, and product should normally ship out within a couple of days or so. Hope that helps.
Hello! We tried PDM medium with 6 clones which had low plasmid yields (less than 50 ng/ul) when grown in LB medium, but unfortunately we obtained similar or lower yields đ . We used QiagenÂŽs Plasmid purification kit