1. Preculture the bacteria overnight in 4ml LB with selective antibiotic.
2. Refresh the precultured bacteria in 20 ml of M9 xylose in flask.
3. Culture the bacteria for 24 hr in normal incubator with 177 rpm and 37℃ condition in 24 hr.
4. Measure the optical density of the sample with 600 nm light wavelength, and then diluted the optical density value to 0.25 ,0.5, 0.75, 1 in 4ml of bacteria respectively.
5. Measure the weight of an empty eppendorf.
6. Transfer 4 ml of bacteria to eppendorf, and then centrifuge them at 16000xg for 3 min.
7. Put the eppendorfs in the 60℃ high-temperature oven with cap opened for 12 hr.
8. Measure the net weight of the bacteria after 12 hr.
9. Analyze the dry cell weight and draw the regression line of it.

M9 medium contains essential salts and nitrogen. It contains 33.9 g/L Na₂HPO₄·7H₂O, 15 g/L KH₂PO₄, 5 g/L NH₄Cl and 2.5 g/L NaCl; suitable for recombinant E. coli strains

• Minimal salts (M9) medium is suitable for non-selective cultivation of E. coli strains for cloning, production of DNA, plasmid DNA and recombinant proteins.
• Suitable for selective cultivation when appropriate antibiotics are added.

Substances		Volume in ml	Volume(M)
M9 salt solution (10X)
	Na2HPO4	100	33.7 mM
	KH2PO4		22.0 mM
	NH4Cl		9.35 mM
	NaCl		8.55 mM
20% xylose		20	0.4 %
1 M MgSO4		1	1 mM
1 M CaCl2		0.3	0.3 mM

1. M9 salt solution (10X)

Na₂HPO₄·2H₂O 75.2 g/L

KH₂PO₄ 30 g/L

NaCl 5 g/L

NH₄Cl 5 g/L

- Dissolve the salts in 800 ml water

- Add water to a final volume of 1 L

- Autoclave for 15 min at 121°C

2. 20% Xylose

- Add 100 g xylose to 440 ml water

- Add water to final volume 500ml

- Autoclave for 15 min at 121°C

3. 1 M MgSO₄

- Dissolve 24.65 g MgSO₄-7H₂O in 87 ml water

- Add water to final volume 100ml

- Autoclave for 15 min at 121°C

4. 1 M CaCl₂

- Dissolve 14.70 g CaCl₂-2H₂O in 94.5 ml water

- Autoclave for 15 min at 121°C

1. Preculture the bacteria overnight in 4ml LB with antibiotic.
2. Refresh the precultured bacteria in 4ml of M9 xylose in test tube.
3. Culture in the normal incubator for 12 hr with 177 rpm
4. Measure the optical density of the sample with 600 nm light wavelength at the beginning (0 hr) , and also measure them after 12 hr. All samples should be measured in triplicate.

DNS solution preparation:

1. Disolve 2.5g of 3,5-Dinitrosalicylic acid (DNS) to 150ml double distilled water.
2. Heat to solution to 45 degree Celsius and add 4g of NaOH. Stir the solution until it is transparent.
3. Add 75g of potassium sodium tartrate and add water to 250 ml.
4. Keep the solution without light exposure. The solution can be used after 7 days.

Principle:

Under base solution, DNS will turn to brown color while reacting with reductive sugar in high temperature. In the specific temperature range, the color will have linear relationship with the reductive sugar concentration.

Calibration:

1. Prepare glucose or xylose water solution to the following concentration: 0, 0.2, 0.4, 0.8, 1.0, 1.2, 1.6, 2 g/L.
2. Take 200 ul of sample, add 200 ul of DNS solution.
3. Heat the sample at 100 degree Celsius for 10mins.
4. Cool down the sample with ice to room temperature.
5. Measure the optical density of the sample with 540nm light wavelength.
6. Draw the graph of sugar concentration with respect to optical density.

Calibration results:

Measurement:

1. Take 200 ul of sample, add 200 ul of DNS solution.
2. Heat the sample at 100 degree Celsius for 10mins.
3. Cool down the sample with ice to room temperature.
4. Measure the optical density of the sample with 540nm light wavelength.
5. Get the concentration of reductive sugar from the Calibration graph.