Protocol for DNA Content Measurement with Hoechst 33342

DNA content determination with Hoechst 33342 Keiko Ookawa-Chinzei

Rev. 1, Oct 4, 2002

For 12-well plate

Tris-NaCl: Dissolve Tris 3.029 g, NaCl 2.92 g, and EDTA 0.93 g in 400 mL of DDW. Adjust pH 7.5, and adjust final volume upto 500 mL with DDW.

Protease stock solution:

Dissolve at 0.05 U/mL in DDW. Aliquot and store at –20 C.

Hoechst 33342 stock solution:

1mM = 5.16 mg in 10 mL Tris-NaCl.

Standard DNA: Dissolve salmon testis DNA (USBiological) at 5 mg/mL in Tris-NaCl. Aliquot and store at –20 C. Use at 1-20 ug/mL.

## Slit width, scan speed must be optimized for your own conditions!

1. Dilute protease stock solution X 1,000 with Tris-NaCl immediately before use.

2. Dilute Hoechst 33342 stock solution X 100 with Tris-NaCl immediately before use.

3. Aspirate the medium from dish, and add (diluted) 2.5 mL of pronase solution.

4. Incubate them at 37 Cin CO2 incubator for 1 hr.

5. Scrape off the cells from dish, and pour cell suspension into centrifuge tube.

6. Sonicate (by “hone”, not “water bath”) and completely homogenize the cells.

7. Aliquot 2 mL of Hoechst solution (diluted) into tubes, and add 200 uL of cell homogenate.

8. Measure fluorescence at 350 nm (slit 2.5) excitation 460 nm emmision (slit 2.5) at scan speed 200.

For calibration:

1. Prepare standard DNA from 1 ug/mL to 20 ug/mL (and 0 ug/mL = blank, too).

2. Aliquot 2 mL of Hoechst solution (diluted) into tubes, and add 200 uL ofDNA standard.

3. Measure fluorescence at 350 nm (slit 2.5) excitation 460 nm emmision (slit 2.5) at scan speed 200.