DNA Quantification
DNA quantification with picogreen (Molecular Probes)
The stock TE buffer (T10E1 pH 7.5) that comes with the picogreen kit is 20x. It has been diluted (25 ml plus 475 ml of Milli-Q), filter sterilized through a 0.2 um filter and frozen in aliquots of 10 ml. Thaw the required number of tubes. When calculating the number of wells that are needed, you should take into account 10 wells of controls (high and low DNA standard curves).
| Number of wells | Amount of T10E1 |
| 100 | 20 ml |
| 50 | 10 ml |
| 25 | 5 ml |
| 12 | 2.5 ml |
The Picrogreen solution is 200x in DMSO. It is in aliquots of 25 ul in amber tubes stored at -20 C. Thaw at room temp and protect the dye from light. Mix the solution in polypropylene tubes (Not glass!). Dilute the picogreen as follows:
| Number of wells | Picogreen concentrate | 1 x T10E1 |
| 100 | 50 | 10 ml |
| 50 | 25 | 5 ml |
| 25 | 12.5 | 2.5 ml |
| 12 | 6.25 | 1.25 ml |
Add solution to black flat-bottomed 96-well plates (Costar #3917). Nunc black polysort strips can also be used (16 wells to a strip; cat # 475523; total well volume 400 ul). The strips can be soaked in 10% bleach , washed, dried and reused.
The lamda-standard is 100 ug/ml. It is divided into 25 ul aliquots. Thaw the standard on ice. For a working solution of 2 ug/ml, the dilution rate is 4 ul plus 196 ul of TE. For a working solution of 50 ng/ml, the dilution rate is 5 ul plus 195 ul of TE. There will be sufficient solution to make the two standard curves below. I use 8 x 1.5ml Epitubes two, labeled 2 ug and 50 ng, and six labeled 20 ng, 2 ng, 1 ng, 500 pg, 50 pg, and 10 pg to make the standards. To make the standard curves, follow the schemes, mix well, and incubate 5 minutes at RT protected from light.
High range standard curve. Use polypropylene tube to prepare dilutions. (All volumes in this table are in ul)
| 2 ug/ml DNA stock | 1 x T10E1 | diluted picogreen | final DNA concentration | Amount of DNA per well |
| 100 | 0 | 100 | 1 ug/ml | 200 ng |
| 10 | 90 | 100 | 100 ng/ml | 20 ng |
| 1 | 99 | 100 | 10 ng/ml | 2 ng |
| 0.5 | 99.5 | 100 | 5 ng/ml | 1 ng |
| 0 | 100 | 100 | blank | blank |
Low range standard curve. Use polypropylene tube to prepare dilutions. (All volumes in this table are in ul)
| 50 ng/ml DNA stock | 1 x T10E1 | diluted picogreen | final DNA concentration | Amount of DNA per well |
| 100 | 0 | 100 | 25 ng/ml | 5 ng |
| 10 | 90 | 100 | 2.5 ng/ml | 500 pg |
| 1 | 99 | 100 | 250 pg/ml | 50 pg |
| 0.5 | 99.5 | 100 | 25 ng/ml | 10 pg |
| 0 | 100 | 100 | blank | blank |
Dilute the unknown DNA in 100ul T10E1 of , add 100ul of the diluted picogreen and incubate for 5 min at RT, rotected from light.
We’ve been using the Wallac 1420 top reader with the fluorescein program at 1.0 or 0.1 sec per well. Build the standard curve to obtain the amount of DNA of your unknown.
Notes: *using the lamda-standard we’ve been able to consistently read down to 250 pg. In order to read 25 pg we’ve had to do the following: The 1 sec/well scan seems to work best. The difference btw the blank and the standard range from ~11 to 30 fluoresecent units depending on the time used. Also duplicates of the 25 pg/ml standard may need to be run to get consistent results. We’ve also been able to quantify fluorescently labeled PCR products (TRFLP, LH PCR etc ) because the quenching effect of the fluorophjore on the primer is minimal when compared to the amount of picogreen bound to the dsDNA.
(last update; Andy Witherell Oct
2004)