PicoGreen dsDNA quantitation assay

C. Tachibana, May 2006 draft (give corrections to Chris)

1. Dilute PicoGreen 200-fold with TE

e.g. 10µl PicoGreen + 1990µl TE

Use a plastic tube.

Make 50µl of diluted PicoGreen for each sample, plus blanks and standards.

Use within a few hours.

2. Make standard(s) of ds DNA (e.g. lambda)

For quick assay the standard should be 100ng/ml ds DNA.

For more accurate readings, make standards that are 100, 25, 10, 2.5 and 1 ng/ml.

3. For each sample or standard, mix 50µl diluted PicoGreen with 50µl sample or standard

Incubate 2-5 minutes, room temp, protected from light.

Try to have samples at about 5-50 ng/ml (so dilute miniprep DNA about 10⁴-fold).

For more accurate readings, do everything in duplicate.

4. Pipet 90µl of samples, standards and blanks into glass cylinder microcuvettes.

Pipet carefully down the side to avoid bubbles, gaps and excessive sample manipulation.

5. Calibrate fluorometer in "blue" channel.

Turn on, warmup for 5 seconds.

Make sure the minicell adaptor in the cuvette holder shows the BLUE label.

Select blue channel with <A/B> button.

Press <STD/VAL> button. Use up and down arrows to set to 100, hit <ENTER>.

Press <CAL> then <ENTER>. Insert blank into the minicuvette adaptor and press <ENTER>.

Insert the 100ng/ml standard and press <ENTER>.

Within 10 seconds of calibration completion, press <ENTER>.

6. Read samples by inserting into the minicuvette adaptor and pressing <READ>

Press <SET>. Select <DISCRETE> and press <ENTER>,

Insert samples and press <READ> to the sample concentration in ng/ml.

Re-read blank and standard at the end.

For more accurate readings, calculate sample concentration using the 1-100ng/ml curve.

Microcuvettes

Turner Designs, www.turnerdesigns.com

100µl cuvettes, 7000-950

PicoGreen Molecular Probes

1ml P7581

1ml kit with TE and control DNA P7589