QUANTIFICATION OF DISSOLVED DNA USING HOECHST 33258
The is a fluorometric method for quantifying DNA. It tends to be useful when organics contaminate DNA extracted from an environmental sample. In this situation a measurement of A260/A280 does not give accurate DNA measurements.
The method is based upon the interaction of the bisbenzimide fluorochrome Hoechst 33258 with the major groove of AT-rich portions of double stranded DNA. Standard curves are generated using calf thymus DNA or salmon sperm DNA
Before starting:
- Clean cuvettes with 10 % HCl. Rinse well (at least 5x) with RO water and then rinse three more times with DI. Alternatively use 15 ml disposable Sarstaedt tubes.
- Dilute standard DNA to 0.1 mg/ml in 1xSSC.
- Make Hoechst 33258 stock (0.6×10-4 M). Then add 1 ml of stock to 100 ml 1xSSC to make a working solution
To make standard curve:
Add the following to a duplicate set of tubes (all in μl):
1xSSC Hoechst 33258 Diluted DNA [DNA] in sample 2000 1000 0 0 2000 1000 5 50 2000 1000 10 100 2000 1000 25 250 2000 1000 50 500 1900 1000 100 1000 1800 1000 200 2000
To quantify unknowns:
Quantify unknowns by adding 1 ml of Hoechst solution to DNA samples and adding 1x SSC to a total of 3 ml.
Determine fluorescence values using:
Excitation = 350 nm Emission = 450 nm
Reference:
Paul, J.H. and B. Myers. 1982. Fluorometric determination of DNA in aquatic microorganisms by use of Hoechst 33258. Appl. Environ. Microbiol. 43:1393-1399