Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
CONTRIBUTOR: The laboratory of Andrew Murray at Harvard University.
PROCEDURE:
1. Grow an overnight culture of E. coli cells carrying plasmid of interest in 50 ml of media (i.e. LB or 2X YT) containing the appropriate antibiotic to select for the plasmid.
2. Centrifuge cells at 6,000 X g for 5 minutes. Decant the supernatant.
3. Resuspend the cell pellet in 1.75 ml of GTE Buffer.
4. Add 0.25 ml of Lysozyme Solution.
5. Incubate on ice (4°C) for 30 minutes.
6. Add 4 ml of fresh SDS/NaOH Solution and mix by inverting the tube several times.
7. Incubate at room temperature for 5 minutes.
8. Add 2.5 ml of Potassium Acetate Solution and mix by inverting the tube several times.
9. Incubate the tube on ice for 10 minutes.
10. Centrifuge the lysate at 27,000 X g for 15 minutes at 4°C.
11. Pipette the supernatant to a new centrifuge tube and discard the cell debris pellet. If the precipitate is carried over with the supernatant, filter the supernatant through siliconized glass wool or Whatman filter paper prewetted with TE.
12. Add 3.6 ml of 100% Isopropanol to the supernatant and mix by inverting the tube several times.
13. Centrifuge the tube to pellet the DNA at 12,000 X g for 10 minutes at room temperature.
14. Decant the supernatant and allow any residual liquid to drip out of tube by turning the tube upside down on top of paper towels.
15. Resuspend the pellet in 2 ml of TE.
16. Add 0.8 ml of 10 M LiCl and chill the tube on ice for 20 minutes.
17. Centrifuge the solution at 12,000 X g for 10 min at room temperature to pellet the RNA.
18. Using a pipette, transfer the supernatant to a new tube and add 1.7 ml of 100% Isopropanol. Mix by swirling the solution in the tube.
19. Centrifuge to pellet the DNA at 12,000 X g for 10 minutes at room temperature.
20. Decant the supernatant as in Step #14.
21. Wash the pellet in 3 ml of 70% Ethanol, centrifuge (as in Step #19), and remove the supernatant.
22. Dry the pellet either by vacuum or air drying at room temperature.
23. Dissolve the dry pellet in 0.2 to 0.5 ml of TE.
SOLUTION:
LB Medium
5 g/liter Sodium Chloride
5 g/liter Yeast Extract
10 g/liter Tryptone
1 ml/liter 1 M NaOH 
70% (v/v) Ethanol
10 M LiCl
TE
10 mM Tris, pH 8.0
1 mM EDTA
Potassium Acetate Solution
2 M Glacial Acetic Acid
3 M Potassium Acetate
SDS/NaOH Solution
1% (w/v) SDS
0.2 M NaOH
Lysozyme Solution
10 mg/ml Lysozyme in ddH2O
GTE Buffer
25 mM Tris, pH 8.0
10 mM EDTA.
15% (w/v) Glucose
YT Medium (2X)
16 g/liter Tryptone
10 g/liter Yeast Extract
86 mM NaCl
REAGENTS AND CHEMICALS:
Ethanol
Tris
EDTA
Glucose
Lithium Chloride
Sodium Chloride
Sodium Hydroxide
Lysozyme
Glacial Acetic Acid
Isopropanol
Yeast Extract
Tryptone
Potassium Acetate
SDS