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Star Republic: Guide for Biologists

Alkaline lysis plasmid miniprep

ChangBioscience.com
1. Inoculate, using a sterile toothpick, 5 ml LB media with antibiotics in a sterile culture tube with a single bacterial colony. For quick screening, cells can be grown in 1 ml of LB media in 1.5 ml eppendorf tubes if the plasmid is a high-copy number plasmid.

2. For screening of insertions, draw grids on the back of a LB plate with a marker pen. Using the same toothpick to replicate the colony on the LB plate within a single square. Label the culture tube and the square with the same number.

3. Using a different toothpick for a separate colony.

4. Grow cells in 37 C incubator overnight with shaking (~225 rpm).

5. For culture tubes, spin down cells using a tabletop centrifuge at 3000 rmp for 5 minutes. Or transfer 1.5 ml bacterial suspension to a 1.5 Eppendorf tube. Spin for 20 seconds in a microfuge. Remove the supernatant.

6. Resuspend cells in 150 ml of P1 (15 mM Tris pH8, 10mM EDTA) + 10 mg /ml RNAse A.

7. Add 150 ml of P2 (0.2N NaOH 1% SDS). Close the caps, mix gently by inverting the eppendorf tubes 3 times.

8. Add 150 ml of P3 (3M KOAc pH 5.5). Mix well, and let sit for 5 minutes.

9. Remove precipitated SDS, proteins, membranes, and chromosomal DNA by centrifugation at 12,000 rpm for 10 minutes.

10. Transfer the supernatant to a fresh 1.5 ml eppendorf tube.

11. Add equal volume of cold phenol:chloroform:isoamyl alchol (25:24:1). Mix well. and centrifugation at 12,000 rpm for 10 minutes.

12. Remove the aqueous (top) phase to a new 1.5 ml eppendorf tube.

13. Add two volumes 95% ethanol. Incubate for 20 minutes on ice or at -20 C.

14. Spin in the microcentrifuge for 10 minutes. Decant the supernatant and wash the pellet once with cold 70% ethanol.

15. Air dry the pellet. Resuspend the pellet in 30 ml TE containing 10 mg /ml RNAse A.