PicoGreen Protocol

PicoGreen Protocol

Items Needed

  • PicoGreen assay kit (ThermoFisher P7589)
  • Clear-bottom black plate (Griener 675096)
  • Molecular-grade water
  • Aluminum foil
  • Access to plate reader (Striepen Lab)

Procedure

  1. Prepare DNA
    1. Thaw, vortex and spin down your DNA plate(s).
    2. (optional) Create a dilution plate for high-concentration samples. Stool is diluted 1:10 - 1:20.
  2. Prepare PicoGreen Reagent
    1. (if needed) Make TE from 20x stock: 2.5 ml 20x TE + 47.5 ml water.
    2. Dilute PicoGreen reagent 1:200 in a foil-wrapped 50 ml tube.
    3. Note: use within a few hours of preparation

      # Plates
      1x TE
      PicoGreen Reagent
      1
      6 ml
      30 ul
      2
      12 ml
      60 ul
  3. Prepare Standard
    1. Create 2 ug/mL lambda DNA standard (if needed, good for 1 month at -20C):
      1. 490 ul TE
      2. 10 ul 100 ug/mL lambda DNA
    2. Make standard curve as follows, if needed (good for 1 week):
    3. *Final concentration after diluted PicoGreen reagent added at 1:1 ratio.

      Std
      Well
      2 ug/ml Std (uL)
      TE (uL)
      [Final] (ng/mL)*
      1
      A1
      250
      250
      500
      2
      B1
      100
      400
      200
      3
      C1
      50
      450
      100
      4
      D1
      25
      475
      50
      5
      E1
      10
      990
      10
      6
      F1
      5
      995
      5
      7
      G1
      1
      999
      1
      8
      H1
      0
      1000
      0
  4. Prepare Plates
    1. Add 50 ul diluted PicoGreen reagent (from step 2) to each well for every sample and standard.
    2. Standards:
      1. Add 50 ul of each standard to wells in column 1 as indicated in step 3b.
    3. Samples:
      1. Add 48 ul TE to all sample wells.
      2. Add 2 ul sample DNA (or DNA dilution) to all sample wells.
  5. Cover plate with foil.
  6. Vortex on slow speed for 10 seconds.
  7. Incubate 2-5 min protected by light.
  8. Read on plate reader at 480 nm excitation.
  9. Export results to Excel document.
  10. Analyze using the Excel analysis template.
CHMI Picogreen Analysis Template.xlsx20.0KB