Version history:
- 4-5-18 - David S.
Purpose: From Jesse Stricker, in RNA lab notebook page 17
Minimal A medium is a defined minimal medium for E. coli growth. It uses (NH4)2SO4 as a nitrogen source. It is somewhat less stringent than M9 or M63 media. MG1655 can grow in it with doubling times of ~80-110 minutes at 37C, depending on the carbon source. DH5alpha does not seem to grow well in minimal A medium
I've used glucose (0.2%), glycerol (0.05%), and succinic acid (0.2%) as carbon sources. 0.2% glycerol appears to inhibit growth of MG1655. Succinic acid gives a lower pH than glucose (6.4 as opposed to 7.0). The cells grow slowly in succinic acid. It's possible that they would grow better in a succinate salt of some sort (Sigma has sodium succinate). It also might be true that you want the succinate ion at 0.2%. Many other carbon sources could be used.
Minimal A medium includes magnesium added from a stock solution. it may also have vitamins added (vitamin B1, also known as thiamine, is often added, and I always add it) to 1ug/L. If amino acids need to be added, they are added to 50ug/L. All three of these don't like to be autoclaved, which is why they're added separately. The vitamins and amino acids are optional, although I always use thiamine, but the magnesium is required.
A few miscellaneous notes on minimal medium are worth writing down. Cultures typically grow to OD600 of 1-2 given 0.2% carbon source. Because their doubling times are so long, sometimes they don't grow all the way into stationary phase in an overnight culture. Lag phase from stationary to log phase is somewhere around an hour or so, but lag phase from rich medium to minimal medium is longer. Induction curves tend to be cleaner in minimal medium, probably because there isn't any nutritional shifting during growth. The cells are often slightly smaller in diameter, which might mean that microfluidic microscopy chips will need to be redesigned.
The recipe given here is taken from J.H. Miller, A short course in bacterial genetics. Minimal media recipes seem to come in any number of variations, so it's worth it to cite the one you use.
1X minimal A medium
- 100mL 10X minimal A salts
- 1mL 1M MgSO4-7H2O
- 100uL 10g/L thiamine-HCl
- carbon source as desired (often 0.2%, 10mL of a 20% stock solution)
- dH2O - to 1L
Sterile filter to sterilize. If you'd rather autoclave, autoclave the minimal A salts and water together, cool, then add magnesium, thiamine, and carbon source. I prefer to use dH2O rather than Milli-Q water in some hope that trace minerals will be provided. This is probably superstition on my part.
1X minimal A medium is 60mM K2HPO4, 33mM KH2PO4, 8.3mM NH4SO4, 1.7mM NaCitrate, and 1mM MgSO4, if my math is right.
Stock Solutions
10X minimal A salts
- 105g K2HPO4
- 45g KH2PO4
- 10g NH4SO4
- 5g sodium citrate-2H2O
- dH2O to 1L
Sterile filter or autoclave
1M MgSO4-7H2O
- 12.3 MgSO4-7H2O
- dH2O to 50mL
Sterile filter
10g/L thiamine-HCl
- 0.5g thiamine-HCl
- dH2O to 50mL
Sterile filter