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Alkaline Lysis Miniprep Procedure
Alkaline Lysis Miniprep Procedure
1. Inoculate a 2 ml culture of LB/amp (50-100 µg/ml) with a single bacterial colony. Place tube in 37°C shaker overnight.
2. Fill an eppendorf tube with approximately 1.5 ml of the culture, centrifuge for 1 minute at 13 000 rpm and remove all liquid.
3. Resuspend pellet in 100 µl of solution I:
Solution I
50mM Glucose
25 mM Tris-HCl (pH8.0)
10 mM EDTA (pH8.0)
4. Add 200 µl of Solution II:
Solution II (freshly prepare)
6 ml: 600 µl 10% SDS
120 µl 10 N NaOH
5280 µl dH2O
5. Mix gently by inverting 4-6 times
6. Add 150 µl Solution III: (3 M K+, 5 M Acetate)
Solution III
5M Potassium Acetate 60 ml
Glacial Acetic Acid 11.5 ml
H2O 28.5 ml
7. Mix gently by inverting 4-6 times
8. Centrifuge at 13 000 rpm for 10 minutes
9. Transfer supernatant to new tube
10. Add 800 µl (2 volume) of EtOH
11. Incubate for 2 miuntes RT
12. Centrifuge at 13 000 rpm for 10 minutes
13. Discard supernatant
14. Add 500 µl of 70% EtOH
15. Centrifuge at 13 000 rpm for 5 minutes
16. Discard supernatant
17. Centrifuge at 13 000 rpm for another 5 minutes
18. Aspirate all liquid, be careful not to disturb the pallet
19. Resuspend pallet in 30 µl DW or elution buffer
| Create Date : 28 มกราคม 2551 |
| Last Update : 28 มกราคม 2551 16:01:10 น. |
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If there is a stage at which an individual life becomes truly adult, it must be when one grasps the irony in its unfolding and accepts responsibility for a life lived in the midst of such paradox. One must live in the middle of contradiction, because if all contradiction were eliminated at once life would collapse. There are simply no answers to some of the great pressing questions. You continue to live them out, making your life a worthy expression of leaning into the light.
-Barry Lopez Arctic Dreams
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