1. Turn on the machine and allow it to warm up for 15-30 minutes; make sure the machine and all solutions are at 23ºC (RT).
2. Place 2 ml of the TNE-dye solution in the cuvette and place the cuvette in the fluorometer.
3. Zero the machine using the zero dial on the fluorometer.
4. Add 1 µl of your DNA standard. For plasmid DNA use the 0.2 mg/L standard that comes with the ABI DNA sequencing kit. Mix the cuvette by hand and place back in the fluorometer.
5. Adjust the scale button until it reads 100 units.
6. Remove the cuvette and empty its contents. Repeat steps 2-5 several times until you get consistent readings. An occasional bad reading is often the result of pipetting error; note that not all the Hancock lab P2 pipetters deliver consistent volumes and this results in readings that can be interpreted as ‘drift’ by the fluorometer. Drift of +/- 5 units can be ignored (i.e. readings of 95 to 105).
7. Repeat steps 2-5 using your own DNA samples. Record the fluorescence units.
8. DNA quantity is calculated as follows: 2 X (your fluorescence units) = DNA concentration in nanograms per microliter.
9. If your DNA concentration is less than 70 ng/µl throw it out and make new template; you will need a significantly higher concentration of DNA if you are using plasmids larger than 10 kb (>150 ng/µl). Using the Qiagen preps and high copy plasmids you should expect your concentration to be in the range of 80-200 ng/µl. I have found it difficult to get consistent DNA sequencing results using Qiagen prep DNA at concentration less than 70 ng/µl.

SOLUTIONS:

10X TNE
For 500 mL solution:

Tris	6.05 g
Na2-EDTA	1.85 g
NaCl	29.2 g

Dissolve above into 400 ml double distilled H₂O and pH to 7.4 with concentrated HCI.  Make to 500 ml.
 

Concentrated dye stock solution

Hoechst 33258 1 mg/ml in double distilled H₂O; store this in the freezer.
 

Working dye solution

1. Add 10 µl of concentrated dye solution to 100 ml 1X TNE
2. Pass the solution through a 0.22-0.5 µm filter.
3. Store this solution in a dark bottle at room temperature for up to 3 months. Note that this solution will go off and you will get inconsistent, or no results at all.