· Stock Dye Solution (1 mg/ml)
Hoechst 33258 (Polysciences, Inc., cat. # 09460) 10 mg
Distilled water 10 ml
Store in the dark at 4°C 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
