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

Prepare fresh daily

? Extended range assay solution (DNA samples: 100 ng/well ? 4000 ng/well)

1 mg/ml Hoechst 33258

Dye stock 100 ml

DPBS 100 ml

Prepare fresh daily

7.Procedure

? Whole samples are washed with PBS and Proteinase K solution is added to cover the samples.The samples are digested over night at 56℃and transferred into a cryotube and can be stored at -20℃.

? Prepare the standards for the extended range assay from the stock solution to obtain a concentration of 4000 ng DNA in 40 μl volume for the first standard.Dilute the standards in DPBS in a row by two down to a concentration of 125 ng in 40 μl.Run standards in duplicates.The 2 blanks consist of DPBS.

or

? Prepare the standards for the standard range assay from the stock solution to obtain a concentration of 500 ng DNA in 40 μl volume for the first standard.Dilute the standards in DPBS in a row by two down to a concentration of 15.6 ng in 40 μl.Run standards in duplicates.The 2 blanks consist of DPBS.

? Pipet 40 μl of standards / samples into designed wells of a 96 well white plate.Run samples in duplicates or triplicates

? Add to each well 160 μl assay solution.

? Tab the plate gently and read the plate at 360 nm (excitation)/ 465 nm (fluorescence emission)

? Prepare a linear standard curve E (465 nm)vs.DNA amount (ng/well)and calculate samples according to their fluorescence values.

Comments

? RNA does not interfere.

? Triton X-100 interferes with the assay

? The DNA sample should not contain ethidium bromide because it quenches the fluorescence of Hoechst 33258.

8.Documentation,Archiving

The original data are stored in a local computer file and as a hard copy in the project log book.

9.Special Requirements,Quality Assessment

10.References

Labarca C,Paigen K.A simple,rapid,and sensitive DNA assay procedure.Anal Biochem 1980; 102:344-352