Pico Green DNA Quantitation
Reagents:
1x TE
Procedure calls for “assay buffer.” Use water if diluted samples will be needed for PCR
Prepare from 20´ concentrate (stored in -20°C freezer)
Make ~1mL for each sample to be analyzed, including duplicates and standards.
Picogreen
Prepare 0.5 mL for each sample to be analyzed, including duplicates and standards.
Mix from 200´ stock stored in -20°C freezer. Make dilutions in plastic as Picogreen can sorb to glass. Cover in foil and store in a cool place. Use within 2-3 hours of preparing
2 mg/mL DNA
Dilute from 50 ´ stock of l DNA stored in -20°C freezer. Amount varies based on standard curve. The table below is for a standard curve set up in a well plate.
Sample Prep:
For soil samples, prepare a 0.01 dilution of extracted DNA (5 mL in 495 mL water). Add 100 mL of diluted sample to each well and add 100 mL of picogreen dye.
Fluorometer Settings:
Turn on the fluorometer and the computer and allow the fluorometer to warm up for 15 minutes before using.
Under “Instrument” and “Read” the settings should be:
Fluorescence
Excitation 480 Slit Width 15
15 Emission 520 Slit Width 20
Integrate time 20 seconds
The local contact for the fluorometer is John Shannon jds1c@virginia.edu
For a pdf of this file click here