User:Andriana/7 June 2012

From 2012.igem.org

I. Continued transformation of BL21 cells

II. Continued and finished Transformation of DH5-α cells:

NOTE: Conduct all procedures aseptically, sterilizing the lips of all containers used

1. Dilute 2 mL of DH5-α cell solution grown overnight in 200 mL sterile LB broth and allow to grow until OD = .5 to .55
2. Transfer all cell-broth solution into 4 polypropylene tubes
3. Keep the tubes on ice for 10 minutes
4. Centrifuge at 2500xg for 10 min at 4C.
5. Pour off supernatant and remove as much LB from the pellet as possible by briefly letting the polypropylene tubes stand upside-down on kimwipes. Sterilize the lips of the tubes afterwards with flame.
6. Resuspend pellets in each of the tubes with about 10 mL of TB buffer and combine solutions into 2 tubes containing about equal volumes. NOTE: If pellets are not dissolving from the bottom of test tubes, try either using a flame sterilized (burn alcohol-wet utensil) inoculating loop or glass stirring rod or sterile pipette tip to dislodge the pellets, but be careful not to contaminate the polypropylene tubes by touching them.
7. Centrifuge the 2 test tubes at 2500xg for 10min at 4C again, draining the supernatant as in step 5.
8. Resuspend the pellets in each of the polypropylene tubes with 10 mL TB buffer, and combine the solutions into one test tube.
9. Add 1.5 mL DMSO and mix
10. Store cells on ice for about 10 min. while putting out the sterile eppindorf tubes (chill on ice)
11. Flash freeze cells with liquid nitrogen and store at -80C.
12. Test:

1. For contamination by plating on ampicillin plates
2. For competency with a transformation

III. Plated BL21 cells and DH5-α cells on ampicillin plates to test for contamination

IV. Made more LB broth and autoclaved stock solutions and glassware