The basics
Preparing Complete Media:
Obtain 500 ml bottle of media.
Add 5.5 ml L-glutamine (1/100).
Add 2.75 ml Pen Strep (1/200).
Add 50 ml FCS for 10% mixture; or add 75 ml for 15% mixture.
Preparing 293 GPG Media:
Obtain 500 ml bottle of DMEM.
Add 5 ml Pen Strep
- 50 ml FCS
- 112 ul Tet (5 mg/ml)
- 560 ul Puro (2 mg/ml)
- 1.7 ml G418 (400 mg/ml)
Thawing Cells:
1. Remove from liquid nitrogen tank.
2. Defrost in 37 degree water bath.
3. Add cells to 10 ml incomplete media.
4. Spin down for 5 min. at 2000 rpm’s.
5. Decant liquid.
6. Add 5 ml of complete media; mix well.
7. Transfer to flask/plate.
8. Incubate.
Trypsinizing Cells:
1. Remove media.
2. Wash with PBS 1X.
3. Add trypsin (~3 ml for large flask).
4. Incubate 1 to 2 min. (Don't leave any longer than necessary!!!)
5. Tap flask if needed to loosen cells.
6. Add media; mix.
7. Transfer to new container.
Harvesting Cells:
1. Discard media.
2. Add PBS (~5 ml for a large plate).
3. Scrape cells loose.
4. Pipet into tube.
5. Spin 5 min. at 2000 rpm’s.
6. Decant liquid.
7. Add 5 ml PBS to tube; resuspend cells.
8. Transfer to round bottom (14 ml) tube if necessary.
9. Spin again; decant liquid.
10. Cap tightly and place in -20 freezer.
