The basics

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Contents

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.

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