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1. Turn on the fluorometer and allow it to warm up for at least 15 minutes.
2. Prepare the amount needed of working dye solution and cover with foil.
CALIBRATION
3. Pipette 2 ml of working dye into the glass cuvette. Wipe the cuvette and insert into the well (always place the
cuvette in the same orientation in the fluorometer) and close the cover.
4. With the ìSCALEî knob adjusted to around 5 clockwise turns from fully counter-clockwise position, adjust the
reading to ì000î with the ìZEROî knob.
5. Add 2 μl of the DNA standard to the cuvette, mix well and re-insert into the fluorometer (in same position).
NOTE: DNA standard should be a similar type (linear, circular, GC%) to the samples to be measured. For
plant DNAs (genomic and inserts) calf thymus DNA is fine. For low DNA concentrations, it is
best to use a standard of 25 or 50 ng/μl. For high DNA concentrations, it is best to use a standard
of 500 ng/μl.
6. Adjust the ìSCALEî knob so that the reading equals the concentration of the standard.
7. Steps 3 – 6 can be repeated once or twice until little change in the reading is observed.
QUANTIFICATION
8. Rinse cuvette with dH20, dry and pipette 2 ml of working dye into the glass cuvette. Wipe the cuvette, insert
into the well (always place the cuvette in the same orientation in the fluorometer) and close the cover.
9. With the ìZEROî knob adjust the reading to ì000î. DO NOT ADJUST THE ìSCALEî KNOB.
10. Add 2 μl of the DNA sample to the cuvette, mix well and re-insert into the fluorometer (in same position).
11. Repeat steps 8 – 10 for each sample.
12. If you have many samples, check the 000 reading every 10-15 samples.
NOTE:

 When the DNA concentrations are very high, it is necessary to make dilutions before reading the
concentration with the fluorometer.

10X TNE Buffer (0.1M Tris-7.4, 10 mM EDTA, 1 M NaCl)

STOCK                                                 500 ml                       1000 ml                   2000 ml
Tris-base (MW 121.10)                    6.05 g                         12.1 g                       24.2 g
EDTA (MW 372.2)                               1.85 g                         3.7 g                          7.4 g
NaCl (MW 58.44)                                  29.20 g                     58.4 g                       116.8 g

Adjust pH to 7.4 with HCl, add ddH2O to make 1 liter, filter to sterilize and to remove any particulate
matter.

1 mg/ml H33258 Dye Stock Solution

STOCK                              10 ml
Hoechst 33258             10 mg
Filtered ddH2O             10 ml

DO NOT FILTER OR AUTOCLAVE DYE SOLUTION.
Wrap tube with aluminum foil and store in refrigerator.
CAUTION: The Hoescht dye is toxic, irritant and carcinogenic ó wear gloves and goggles when handling and
use extra precaution.

Working Dye Solution

STOCK                                            50 ml                             100 ml
1 mg/ml Dye Stock                    10 μl                                20 μl
10X TNE                                          5 ml                                10 ml
Filtered ddH2O                             45 ml                             90 ml