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
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.