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. 

 

DNA

ng/mL

2 mg/mL DNA

l mL

TE

mL

Picogreen

mL

0

10

50

100

200

500

0

1

5

10

20

50

100

99

95

90

80

50

100

100

100

100

100

100

 

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

                          Emission 520                    Slit Width 20

                          Integrate time 20 seconds

 

The local contact for the fluorometer is John Shannon jds1c@virginia.edu

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