Mullins Molecular Retrovirology Lab

A+B

1. Fill appropriate number of 96-well plates with 110 µL LB-Amp.

2. Use 96-pin sterile replicators to replicate each original into 2 new plates, A and B.

3. Make sure rows and columns are aligned correctly (this is NOT obvious!).

4. Grow o/n (~ 20 hrs) at 37 °C in humid chamber (wet paper towels in container covered loosely).

5. Log clones that did not grow on appropiate sheet.

6. Include number of plate, date, initials.

7. Seal plates with aluminium foil.

8. Spin plates 10 min at 2000 rpm.

9. Using Transtar 96-well pipet, take off supernatant.  Wash Transtar cartridge between aspirating plates as follows: 
   Set up 3 pipet tip box lids; one filled with 10% bleach, two with sterile water.

   1. Pipet up/down in 10% bleach.
   2. Rinse by pipetting in first container of sterile water.
   3. Rinse again in second container of sterile water.

10. Add 100 µL LB-Amp/15% glycerol using clean Transtar apparatus.

11. Seal plates with aluminium foil.

12. Vortex briefly to resupend pellet.

13. Store at -80°C, original and plates A for Bumgarner, plates B for Katze/Mullins.

C

1. Fill appropriate number of 96-well plates with 110 µL LB-Amp.

2. Thaw plates B for ~10 min.

3. Spin plates 1 min at 2000 rpm.

4. Use 96-pin sterile replicators to replicate each plate B into 1 new plate, C.

5. Grow o/n at 37°C in humid chamber.

6. Log clones that did not grow on appropriate sheet.

7. Include number of plate, date, initials.

8. Seal plates with aluminium foil.

9. Store at -80°C, plate C for Katze/Mullins