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
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”
I tried this medium and got a whopping 6.5x increase in plasmid DNA production compared to LB! Highly recommended!!
Nice, I am glad it worked
Simple idea behind PDM is that the chemicals in the media “react” in the cell to form complex biostructures. They calculated the optimal concentration of each ingredient that would give you a high biomass and lots of plasmid. When you are isolating plasmid, you want your cells to divide like crazy without worrying about spending their energy on transcription or translation, obviously aside from the required amount to sustain cell growth. Hence, PDM would probably suck for protein isolation, although LB or TB can be used for this purpose because of the fact that they are not optimized for constant cell division and low protein synthesis.
About the autoclaving: Glucose will not brown unless heated with amino acids (called a Maillard reaction) as Liam said. Phosphates should be autoclaved separately as usual.
With this medium, I have successfully isolated 60-70 mg pure high-copy plasmid from 1L cultures (no kits involved).
I made a 10x solution of KH2PO4 + Na2HPO4, 10x Glucose, and a 1.2x of the rest. I autoclaved them separately, but when I mixed together something salted out and won’t dissolve, even upon boiling. I’ll repeat to once more to make sure I didn’t screw up the weighing, but this should work right?
Can anybody comment about purity of the plasmid? I mean, all plasmid preparation protocols are adjusted for defined portion of bacterial mass, and if this media increase amount of bacterial cells – one will get excess of material => incomplete lysis etc…
Hi guys
Yes, sometimes something does not dissolve or precipitates out, and I am not sure which component it is. I suspect it is the phosphate salts, and so I generally cool everything to room temperature or lower before combining, and keep a stirrer in the bottle to make sure everything mixes properly. It seems to happen more often with agar plates, so I cool everything in a 55oC waterbath before combining, although the buffer and glucose is alot cooler than that by the time it is added.
Ammonium chloride, potassium phosphate and the Magnesium salt react together and form Ammonium Magnesium Phosphate which is slightly soluble in water. The concentration of the Ammonium Magnesium Phosphate is near its solubility point (0.01%) and could percipitate.
The same could happen with carbonate dissolved in the water and magnesium salt.
Hi,
in the Michael Danquah and Gareth Forde`s paper there is a medium referred to as PDMR, better than PDM, that is the PDM with optimized C:N ratio through variations in the concentration of NH4Cl. In the paper don`t appear the recipe for PDMR, or the C:N ratio of the PDM to infer the addition of NH4Cl
Someone have a hint about this?
hi there,
anyone try this media for recombinant protein expression? if so, how did it fare compared with lb?
S
Hey Sean!! It’s Catherine from the Margolick lab!! Thought I recognized the dreads… Just sent a “friend request”. Haven’t tried the media I’m afraid…
I have red all the good comment about this medium.
I have tried this PDM medium for miniprep and I was really desapointed, my DNA concentration was divide by two;I made the mistake of mix all the ingredients together and after that sterilize the liter of media.
Have you any ideas or protocols to fix this bad rates of DNA purification.
What is the good way to use this media lease it’s really important for me.
This is my common protocol for miniprep
one colony in 5 ml of LB +antibiotic
Growth overnight
spin down
Qiagen miniprep kit
elution in water 50 uL
I tried PDM and the result is decrease by two of the DNA cocnentration.
Have you any idea about That
Has anyone tried to modify this for protein expression? I expect there to be some issues with the glucose concentration if you’re using an ara-driven protein expression vector. What about using glycerol as the carbon source?
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 :-( . We used Qiagen´s Plasmid purification kit
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 :-(
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.