Team:NCKU Tainan/Protocol

Protocol

Dry Cell Weight

Dry Cell Weight ×

1. Preculture the bacteria overnight in 4ml LB with 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 opening for 12 hr.
8. Measure the weight of eppendorfs after 12 hr.
9. Analyze the dry cell weight by making a regression line with pre-test and pro-test of the weight of eppendorf.

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Minimal Salts (M9) Medium Xylose Preparation

Minimal Salts (M9) Medium Xylose Preparation ×

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
	NaCl		8.55 mM
	NH4Cl		9.35 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

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PRK Toxicity Test

PRK Toxicity Test ×

1. Preculture the bacteria overnight in 4ml LB with antibiotic.
2. Refresh the precultured bacteria in 4ml of M9 xylose in flask.
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 in triplicate.

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