Yeast FACS_生物行

Wednesday, May 05, 2004

Description
Yeast FACS

Procedure
1. Grow cells to less than OD = 1 (i.e < 2 x 107 cells/ml)

2. Spin down approx 500 ul. Amount of cells should be about 5 to 10 ul volume. Too many cells does not work!

3. Resuspend in 300 l and dropwise add 700 l of 95% EtoH.

4. Incubate cells on ice for 2-24 hours.

5. Spin down hard for 5 min, wash in 1 ml 50 mM Citrate buffer pH7.4 to get rid of ethanol and resuspend in same buffer.

6. Sonicate for 10 s at setting 30% to remove clumps (you may need to do a trial and check under a microscope).

7. Spin down again hard for 5 min and resuspend cells in 1 ml of 0.25 mg/ml RNase in citrate buffer (pH 7.4).

8. Incubate for 2 h at 50 degrees C.

9. Add 50 l of 20 mg/ml Proteinase K (from stock dissolved in dH2O stored @ 20 degrees C).

10. Incubate for 2 to 2.5 h at 50 degrees C

11. Add 1 ml of 16 g/ml propidium iodide in 50 mM NaCitrate (made from 100x propidium iodide stock stored @ 20 degrees C). Cells may be sonicated again if required.

12. Let stand at r/t for 30 min. Keep away from light. Read on FACS machine or store for 1 week at 4 degrees C.

1 M Citrate buffer stock - Make 1 M NaCitrate. Carefully adjust the 50 mM dilution with a few grains of citric acid crystals to reach Ph 7.4. Store at room temp.

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Yeast FACS