1.Safety precautions

Hoechst 33258 is carcinogen and toxic.Use with adequate ventilation.Avoid contact with eyes,skin,clothing.Wash after handling.Keep container closed.

2.Rationale

For biochemical quantifications it is important to normalize the parameters to the cell number or the DNA content of the sample.

There is a simple and rapid assay for quantitative DNA determination in cell or tissue samples.The method is based on the enhancement of fluorescence seen when bisbenzimide (Hoechst 33258)binds to the minor groove of DNA .Crude homogenates in which chromatin has been dissociated with high salt buffer can be assayed directly and reliable in a few minutes.The dissociation of chromatin is critical to accurate determination of DNA in biological materials.

The fluorescence of Hoechst 33258 is related to the AT content of a DNA sample,so it is very important to use a standard similar to the sample under investigation.The calf thymus DNA standard is double-stranded,highly polymerized,and it has an approximate content of 60% AT (40% GC).

3.Application

To be performed by laboratory personnel and persons trained in practical biochemistry after qualification by an instructor.

4.Applicable documents

PE HTS 7000 Bio Assay Reader instructions

5.Definitions

no special

6.Material

・ PE HTS 7000 Bio Assay Reader

・ Two filters to provide an excitation wavelength of approximately 360 nm with a band with of 100 nm and to detect the emission at 465 nm

・ 96 well white plates

・ DNA standard

Calf Thymus DNA Sigma ‘ultra pure’,cat.# D-4764 (20 units º 1 mg DNA )

Dissolve DNA in DPBS (see below)to 100 µg/ml,stirring over night at 4℃

・ Proteinase K ,Roche cat.# 1 000 144 0.5 mg/ml in phosphate buffer containing

10.68 g/l NaH2 PO4 2H2 O,8.45 g/l Na2 HPO4 7H2 O and 3.36 g/l Disodium-EDTA in Milli-Q water.pH is adjusted to 6.5.

・ Phosphate buffered saline,pH 7.4 containing 2M NaCl (DPBS )

・ Stock Dye Solution (1 mg/ml)

Hoechst 33258 (Polysciences,Inc.,cat.# 09460)10 mg

Distilled water 10 ml

Store in the dark at 4℃ for up to 6 months,do not filter

・ Standard range assay solution (DNA samples: 10 ng/well- 500 ng/well)

0.1 µg/ml Hoechst 33258

Dye stock 10 µl

DPBS 100 ml