Hewson Lab at Cornell


Team Aquatic Virus - Microbial Oceanography, P.I. Ian Hewson, Ph.D.

Pico Green Quantification of DNA by qPCR Machine

Hewson Lab DNA Quantification protocol

Modified from original protocol by Peter Countway, USC.

Supplies:

  1. PicoGreen quantitation kit (# P-7589) from Molecular Probes, which includes:
    1. Component A:  1ml PicoGreen dsDNA quantitation reagent in DMSO
    2. Component B:  25 ml 20x TE buffer (200mM Tris HCL, 20 mM EDTA, pH 7.5)
    3. Component C:  1 ml l-DNA Standard (100 mg/ml in TE buffer)
  2. Pipettes:  1ml, 200 ml, 100ml, 10ml, 2µl
  3. Nuclease-Free Water (Ambion)
  4. (2) sterile, 1.5 ml eppendorf tubes, one for TE working solution, and one for PicoGreen working solution.
  5. (2) sterile, 1.5 ml centrifuge tubes for l-DNA, one for primary dilution, and one for secondary (working) solution.
  6. 96 well PCR plate or 8-tube strips of optical quality PCR tubes + caps.

Notes before starting.

  1. Work fast. This is time sensitive, especially the Pico addition part!
  2. Keep the Pico in a drawer while you make dilutions of standards, and your samples. This is important.
  3. Use a qPCR rack
  4. When closing tubes make sure the lids are on ALL the way on ALL tubes. You can pick the strip up and push down.
  5. Spin down the tubes after sealing with caps.

Prepare the following:

  1. 1 x TE Buffer (1.2 ml) Pipette 60 ml of 20x TE buffer from the kit into a 1.5 ml centrifuge tube.  Add 1.14 ml nuclease free water.
  2. PicoGreen Reagent (0.6 ml) Prepare a 20-fold dilution of the stock PicoGreen.  Pipette 597 µl of 1x TE buffer (prepared above) into a 1.5 ml centrifuge tube.  Add 3 ml of PicoGreen Stock.  Note:  the PicoGreen is a mixture containing DMSO, which is a solid below 18°C.  The stock PicoGreen should be centrifuged after it melts to remove any liquid from the cap before opening the micro-centrifuge tube.  The PicoGreen should be kept out of direct light in a drawer.
  3. l-DNA Standard (40 ml) Pipette 39.4 ml of 1x TE buffer, add 0.6 ml of stock l-DNA (100 mg/ml) into an eppendorf tube, mix by inversion.  This solution is 1.5 mg/ml l-DNA.  Mix 10 ml of the 1.5 mg/ml l-DNA with 90 ml 1x TE buffer to get 1 ml of “working” standard.  Make standard curve dilutions from this (see attached dilutions).
  4. Prepare a standard curve, pipetting 1x TE then DNA into wells of the 96-well plate.  Generally, 0.5 ml of sample (DNA concentrate) plus 14.5 ml of 1x TE is combined for unknowns.
  5. Add 15 ml of PicoGreen reagent to wells for a total volume of 30 ml in each well and cap.  Keep samples/standards in the dark, then read after 5 minutes in qPCR machine using the Plus/Minus setting...

TE Buffer

DIW (ml)

Stock (ml)

Concentration

Total (ml)

TE Stock (20X)

 

 

20X

 

TE Working (1X)

1140

60

1X

1200

Pico-Green Rgt

Dilution

Stock (ul)

1 X TE (ul)

Total (ul)

Pico-Grn Stock

1

 

 

 

Pico-Grn Working

0.005

3

597

600

Lambda DNA

ng/ml

Stock (ul)

1 X TE (ul)

Total (ul)

λ DNA Stock

100000

 

 

 

λ DNA Primary

1500

0.6

39.4

40

 

ng/ml

Primary (ul)

1 X TE (ul)

 

λ DNA Secondary

150

10

90

100


Standard Curve

Samples (example 3 samples)

Conc. à
ng DNA/ml

150 ng/ml
DNA Stock (ul)

0.005
Pico-Grn Rgt. (ul)

1 X TE (ul)

Total Volume (ul)

0.0

0

15

15

30

50.0

5.0

15

10

30

75.0

10.0

15

5

30

150.0

15.0 (1o Stock)

15

0

30

750.0

10.0 (1˚ Stock)

15

5

30

1500.0

15.0 (1˚ Stock)

15

0

30


Conc.
ng DNA/ml

DNA Sample (ul)

0.005
Pico-Grn. Rgt (ul)

1 X TE (ul)

Sample 1

0.5

15

14.5

Sample 2

0.5

15

14.5

Sample 3

0.5

15

14.5

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