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Team:Warsaw/Media
From 2010.igem.org
Alkaline lysis
1. Grow single colony in 5ml of LB (+ ampicillin)
2. 1.5 ml into eppendorf.
3. Pellet cells at 5 000 rpm for 3 min.
4. Discard supernatant, add 1.5 ml of culture and pellet again with the same parameters.
5. Discard supernatant, leave pellet as dry as possible.
6. Resuspend pellet in 200 μL buffer I. Put on ice and wait for 5 minutes.
7. Add 400μL freshly prepared buffer II, mix by inverting 5-6 times(DO NOT vortex!)
8. Put on ice and wait for 5 minutes.
9. Add 200μL buffer III, invert tubes to mix. Incubate on ice for 5 min.
10. Centrifuge at 12 000 rpm for 10 min.
11. Carefully take the supernatant into a new tube.
12. Add 480μL of cold isopropanol. Invert eppi several times.
13. Centrifuge at 12 000 rpm for 10 min at low temperature.
14. Rinse the pellet with 1ml 70%EtOH.
15. Centrifuge at 12 000 rpm for 10 min at low temperature.
16. Air dry the pellet.
17. Dissolve each in 60μL TE with RNAse.
