Protocol for preparation of minimal media
1. Preparation of stock solutions
A. Vitamin Solution (1000 ml)
1.1 mg biotin Store in the refrigerator
1.1 mg folic acid* Store at room temperature
110 mg PABA (para-aminobenzoic acid) Store in the refrigerator
110 mg riboflavin* Store at room temperature
220 mg pantothenic acid Store in the refrigerator
220 mg pyridoxine HCl* Store at room temperature in dessicator
220 mg thiamine HCl* Store at room temperature
220 mg niacinamide Store at room temperature
* Note that these vitamins are light sensitive.
Add 500 ml H2O and 500 ml high-purity ethanol to the vitamins and then filter sterilize.
The solution will be bright yellow. Store the vitamin solution in the refrigerator in a dark
bottle (we wrap our solution with aluminum foil).
Proper storage for each of the individual vitamins is shown to their right.
B. Metal Stock Solution (100 ml)
8 ml concentrated HCl
5 g FeCl2·4H2O
184 mg CaCl2·2H2O
64 mg H3BO3
40 mg MnCl2·4H2O
18 mg CoCl2·6H2O
4 mg CuCl2·2H2O
340 mg ZnCl2
605 mg Na2MoO4·2H2O
Bring to a volume of 100 ml with H2O. Initially the solution will be green and you will
need to stir for several hours before everything dissolves. Store the metal stock solution
at room temperature.
C. "O" Solution (500 ml)
Add 10 ml Metal Stock Solution (above) to 26.8 g MgCl2·6H2O then add H2O to 500 ml.
Filter sterilize and store at room temperature.
Note that the Metal Stock Solution is only used to prepare the "O" solution and is not
added to the minimal media.
D. "SBMX" Solution (500 ml)
To 16.5 g KH2PO4, 87.5 g K2HPO4, and 18.25 g NaCl add H2O to 500 ml. Autoclave and
store the "SBMX" solution at room temperature. The pH of this solution should be 7.5,
however if done correctly no pH adjustment should be necessary.
E. "S" Solution (100 ml)
To 4.8 g K2SO4 add H2O to 100 ml. Autoclave and store the "S" solution at room
temperature.
F. Thiamine Solution (Small volume)
Make up a 1 mg/ml solution of thiamine in H2O. Store the thiamine solution in the
refrigerator. We generally make smaller stock solutions of the thiamine solution to avoid
letting the solution get to old.
2. Preparation of 1 liter of minimal media
To create the growth media add together
940 ml H2O
40 ml "SBMX" solution
1 ml "S" solution
Autoclave and cool
Mix together
2 ml "O" solution
1 ml vitamin solution
1 ml thiamine solution
Filter sterilize the mixture and add to cool growth media
Add together
1 g 15NH4Cl or (15NH4)2SO4
5 ml H2O
Filter sterilize and add to cool growth media
Add together
4 g glucose
10 ml H2O
Filter sterilize and add to cool growth media
Add antibiotic of choice
Note: We have found the most cost effective amount of glucose to add is between 3 and 4 grams.
3. Cell Growth
• A 5 ml culture of LB (10 g Bacto tryptone, 5 g Bacto yeast extract and 5 g NaCl per liter)
is inoculated using a single colony from a fresh LB plate and grown for ~ 6 hours at 37º
C with shaking; they should reach on OD600 of 0.5 - 1.0.
• Use 0.2 ml of the LB culture to inoculate a 25 ml minimal media culture and grow
overnight at 30 - 37º C with shaking; they should grow to an OD600 of 2-3.
o Note: Use 25 ml starter culture per liter. Therefore, if more than 1 liter of minimal
media will be induced the starter culture size will need to be increased.
• Spin down the cells (~2000g) to remove supernatant and resuspend cells in 10 ml of
minimal media.
o Note: We often skip this spin down step and simply inoculate the 1 liter of
minimal media culture with the 25 ml overnight starter culture.
• Use the 10 ml of the resuspended cells from the overnight starter culture to spike 1 liter
of minimal media culture. Grow and induce cells as normal.
Reference: Weber DJ, Gittis AG, Mullen GP, Abeygunawardana C, Lattman EE and Mildvan
AS. "NMR docking of a substrate into the X-ray structure of staphylococcal nuclease."
Proteins (1992) 4, 275-287.
Special Notes:
• The original Weber paper used two additional solutions. One is a T-U solution of
thymine and uracil and the other is the "M" solution which contains MOPS buffer and
tricine. These both have been dropped in the protocol we now use. I am not positive but
the removal of the "M" buffer was likely done to remove a possible carbon source for the
bacteria to use.
• Glucose can cause some repression of IPTG inducible transcription. Therefore, if you are
inducing at low cell densities OD600 < 0.8 where significant glucose may be present a
higher IPTG concentration may be needed.
• In general it is a good idea to try growing cultures to different OD600 values, inducing for
various times and with various amounts of IPTG and to try expression at lower
temperatures such as 30º C. All of these parameters can be adjusted to maximize protein
expression and solubility.
from http://structuralbiology.uchc.edu/Minimal_media.pdf
