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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PURIFICATION: EXTRACT FROM CELLS

PREPARATION OF CLEAR LYSATES FROM BACTERIA

Preparation of Clear Lysates from Bacteria
Contributor: The Laboratory of Giovanna Ferro-Luzzi Ames at the University of California, Berkeley
 
Procedure
1. Grow a 50 ml bacterial culture in LB overnight.

2. Centrifuge to pellet cells (approximately 1,500 X g) and discard the supernatant.

3. Resuspend the pelleted cells in 500 μl cold Tris Buffer 1.

4. Transfer cell solution to a thick walled Pyrex tube.

5. Add 100 μl of Tris Buffer 2

6. Incubate on ice for 10 min.

7. Add 200 μl 250 mM EDTA

8. Incubate on ice for 10 min.

9. Add 10 μl of RNase A and 10 μl of RNase T solution.

10. Incubate on ice for 10 min.

11. Add 800 μl of Triton Solution.

12. Incubate on ice for 10 min.

13. Centrifuge at 18,000 X g for 45 min at 4°C.

14. Remove the clear lysate using a Pasteur pipette.

15. Store the lysate at -20°C or -80°C for long term storage.

Solutions
RNase A   1 mg/ml boiled RNase A
250 mM EDTA
Tris Buffer 2   250 mM Tris, pH 8.0
Prepare just before use
10 mg/ml Lysozyme
Tris Buffer 1   25% (w/v) Sucrose
50 mM Tris, pH 8.0
Luria Broth (LB)   5g/liter Yeast Extract
10g/liter Tryptone
1ml/liter 1 M NaOH
5g/liter NaCl
1 M NaOH
Triton Solution   50 mM EDTA
50 mM Tris, pH 8.0
0.1 % (w/v) Triton X 100
RNase T   1 mg/ml RNase T1
 
BioReagents and Chemicals
Sucrose
Triton X-100
Sodium Hydroxide
EDTA
Lysozyme
Yeast Extract
Tris
Tryptone
Sodium Chloride
 
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