Hoechst 33258 DNA assay

PBE buffer

100 mM Na₂HPO₄ (7.1 g/500 ml)

10 mM EDTA (1.86 g/500 ml)

pH 6.5

Papain solution

125 ug/ml papain (Worthington Biochemical; 0.1 ml of 25 mg/ml stock per 20 ml)

10 mM cysteine (0.035 g/20 ml)

in PBE buffer

10X TNE solution

12.11 g Tris base

3.72 g EDTA

116.89 g NaCl

in 1 L total diH₂O

pH 7.4

filter through 0.45 um

store at 4°C

Assay solution

10 ul of 1 mg/ml Hoechst 33258 (diluted in 1X TNE)

10 ml 10X TNE

90 ml diH₂O

Hoechst 33258 DNA assay

• 1. Prepare working calf thymus DNA solution at 10 ug/ml by adding 10 ul of 1 mg/ml DNA stock solution to 990 ul of 1X TNE.

  2. Prepare the following standards in individual wells of a black 96 well plate:

ul DNA working solution ul 1X TNE resulting ug DNA

0 100 0.0

10 90 0.1

20 80 0.2

30 70 0.3

40 60 0.4

50 50 0.5

• 1. To other individual wells, add 4 ul of digested sample and 96 ul of 1X TNE.

  2. Add 100 ul of assay solution to each sample and standard well.

  3. Read each sample on fluorometer (360 nm/ 465 nm) by selecting Hoechst DNA assay on the flourometer and loading your plate.

  4. Prepare standard curve and calculate DNA in sample wells, taking dilution into account.