- Get 2 X 9 epi tubes and label 1-9

- Chill 4, 6, and 8 since they are the ones to be harvested at this point.

- Need cold PBS or CPBS

- 3 Scrapers

- P-1000 & P-200 pipette w/tips.

1. W/P-1000 pull off media. DO NOT ASPIRATE.

2. Add 1 ml of PBS to each well. Rock it, don't want the well to dry out.

3. Scrape cells. Pull; don't push to prevent skipping of scraper. Tilt the wells so that you can see the cells on the plate better.

4. Grab bucket w/epi tubes, place in hood.

5. Pipette up the PBS w/cells, rinse to make sure you got all the cells.

6. Take to cold room and centrifuge in the swinging bucket centrifuge @ 2000 rpm for 5 min.

7. Pipette out PBS supernatant (come in w/P-1000 & then w/P-200 for last little bit)

8. Resuspend in 200 µl of TX Lysis buffer w/DTT & PIC

9. Incubate on ice for 20 min, vortexing every 10 minutes

While waiting put bleach into wells and throw in incinerating biohazard bag.

In the last 5 minutes label w/Name & date the second set of 1-9 epi tubes.

Put in ice #'s 4, 6 and 8. This is to chill the epi tubes since they will be used soon.

10. Centrifuge in the fixed angle centrifuge in 4_C walk-in @ top speed for 5 minutes.

11. Pull off supernatant & transfer to new tubes.

12. Place in -20_C freezer

13. In 3 Hours repeat process for the remaining epi tubes, making changes as needed.