I often wonder why it is that molecular biology researchers stubbornly refuse to change 40 year old methods that, while they 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.
But by adding in a few more ingredients or being more economical on others (especially yeast extract and tryptone) that you could get a higher plasmid yield, quicker and with less money.
To counter the naysayers, nobody wants to make very complex 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 being that 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 , 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 compared to 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
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Liam Thompson
Hi,
Glucose, KH2PO4 and Na2HPO4 are sterilized by autoclave.
Have I to autoclave the others ingredients, or by filtration?
Ivonne
You sterilise everything by autoclaving, but the glucose in a separate bottle, and the phosphate buffers in a separate bottle. When combining them all together, they don’t autoclave so well, but separately they’re fine.
Good luck
Not sure what the idea is here. Is the medium facilitating more rapid E. coli growth, resulting in greater cell density and therefore higher plasmid yield?
How much of the PDM podwer should be added to 1 liter of diH2O?
Hi Paulo
The medium is for 1L of medium.
CK
As I understand it, it allows greater cell densities to be reached, and the buffering and salts allow for greater plasmid stability, the glucose allows for quicker cell growth. Apart from that, I don’t think there is much more,
Hi to all,
I tried with the above mentioned PDM media to grow my plasmid. Trust me, this media will really work very efficiently for the maximum production of cell density.
Good work!!!!!!!!!!!!!!1 Keep it up
Thanks Sudheer, you pimp that media.
Cheers
Normal growth time for LB is about 15-17.5 hours. In the paper presented by Danquah and Forde, it displays 12 hour growth as approximately the best growth time when using PDM. Is this what others have used?
Looks promising based on the feedback here. The lazier option: inoculating your LB overnight 3 hours earlier than usual?
One point : Glucose + autoclave = Caramel!
You should always filter sterilise glucose.
Sure you can filter glucose, but as long as it is autoclaved with yeast extract or another amino acid source, there will be no caramel, I’ve done it plenty times and never had a hassle.
Sorry, that’s meant to read “without”