M9 minimal media - its not working! - (Jun/08/2007 )

Below is my recipe for M9 minimal media and for some reason it will not support growth (even the controls)! It is incredibly fiddly and time consuming to make – does anyone have any ideas where I am going wrong? …….or have a better method?
Thanks in advance…..
Evejoss

M9 minimal media
Composition per litre:
Na2HPO……………………..6.0g
KH2PO4………………….….3.0g
NH4Cl ………………………1.0g
NaCl…………………………0.5g
Carbon source solution….….10ml
MgSO4.7H2O solution…….....1ml
Thamine HCl solution…..…...1ml
CaCl2 solution…………….…1ml

Glucose solution / carbon source per 100 ml: 20g in 100ml - Make 2g in 10ml
D-Glucose ……………….…...20g thus 2ml c-source into 198ml medium

MgSO4.7H2O solution per Litre: 246.5g in 1000ml
MgSO4.7H2O………….…...246.5g 24.65g in 100ml

Thamine HCl solution per 10ml: 10mg in 10ml (1000mg in 1g)
Thamine HCl………….…...10mg 100mg in 100ml (100mg = 0.1g)

CaCl2 solution per litre: 14.7g in 1000ml
CaCl2………….…..………..14.7g 1.47g in 100ml

Aim
To make five 200ml bottles of M9 minimal agar each containing a different carbon source  to pour plates with.

Preparation:
1. Dissolve agar in 800ml dH2O (aliquot into 5x160ml) -->autoclave.
2. Equilibrate water bath to 55oC
3. Make up separate solutions (carbon source, MgSO4 and CaCl2) -->autoclave.
4. Make up thiamine solution and filter sterilise.
5. Make up x 10 salts in 1 litre (aliquot in to 10x100ml) --> autoclave.
6. Make up antibiotic solution (50mg/ml) --> filter sterilise.
7. Make up carbon source -->autoclave.

Cool agar bottles to ~55oC
1. Add 200μl of MgSO4 solution, 200μl of CaCl2 solution, and 200μl of thiamine solution to a sterile test tube.
2. Add 20ml sterile x10 salts to each agar bottle.
3. Add contents of test tube to agar/salts.
4. Add 200μl of ampicillin to medium.
5. Add 2ml of carbon source.
6. Shake well. --> pour plates…

M9 minimal media can be tricky... I had to make it multiple times before getting it right. I'm not sure but you might want to re-check your recipe as I used much more thiamine HCl and much less CaCl2. After checking on the internet, there seems to be quite a bit of discrepancy amoung recipes...makes it hard to figure out what is correct. This may seem silly but we all make mistakes like these...are you sure you are making the 10X M9 salts? The recipe given at the top of your post is the final 1X concentrations. Also, my recipe called for 50ml of the 10X salts for only half a liter... that's a lot more than your recipe calls for. The only other thing I can think of is that the order in which everything is mixed together is important. Hope this was at least a bit of help.

thank you! i will keep trying...........