Protocol for Preparing pKO3/pKOV

Day 1:

1. Take an LB/chloramphenicol (20 ug/ml) plate from the cold room and allow it to come up to room temperature. (~30 min).
2. Locate the pKO3, pKOV stains in the -80^oC freezer. (On last check on 8/21/00, it was in Dereth's "Most Requested Strains" box lying unracked on the bottom shelf of the -80^oC. Many lab members also have their own frozen stock). (Keep on dry ice if streaking at your bench. Or...)
3. Bring labeled plates and streaking tool to freezer area. Streak straight from frozen stock.
4. Store plate upside down in 30^oC incubator overnight.
5. Make sure you have small LB/chloramphenicol vials ready for stabbing tomorrow. Have however many vials you need to prepare cultures for.

Day 2:

6. Check your plate from Day 1 - make sure colonies have grown. pKOV grows a little slower than pKO3.
7. Remove LB/chloramphenicol vials from cold room and allow to come up to room temperature.
8. Wipe down bench with alcohol.
9. Turn bunsen burner on to low flame.
10. Clean your stab-picking tool (eg., platinum wire) by wiping it down with alcohol, heating in flame.
11. Pick a small, isolated colony and stab it 3 times in the agar in the vial. Use a triangle-shaped pattern. Repeat for all the colonies you want to pick.
12. Wipe down the picking tool after each pick.
13. Place vials, loosely capped, in the 30^oC incubator overnight.

Day 3:

14. Check vials to make sure your stabs have grown.
15. Label each vial with name of strain, the date, and your initials. Also label top of tube with small, circular label.
16. Wrap the edge of the cap with a small piece of Parafilm.

Note: pKO3 and pKOV (formerly pKO5) are the most popular strain requests. We sometimes receive requests for other strains - most of these are kept in the "Most Requested Strains" box in -80^oC freezer. You can check the old -80^o green binder or the new -80^o maroon binder directory for any others. Only pKO3 and pKOV need to grow in LB/chloramphenicol. Most others can be grown on LB only.