Team:Calgary/Notebook/Protocols/picogreen

From 2012.igem.org

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TITLE=PicoGreen Assay Protocol – 1/100 Assay|
TITLE=PicoGreen Assay Protocol – 1/100 Assay|
CONTENT = <html>
CONTENT = <html>
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<p>This protocol describes a method of quantifying the amount of DNA in a given sample by fluorescence of the PicoGreen dye when it intercalates in the DNA.</p>
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<h2>Materials</h2>
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<ul>
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<li>Plate reader</li>
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<li>Black 96-well plate</li>
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<li>15mL Falcon tubes</li>
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<li>Aluminum foil</li>
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<li>Ice bucket and ice for DNA samples</li>
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<li>PicoGreen dye</li>
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<li>20x TE buffer</li>
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<li>DNase free water</li>
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<li>DNA samples</li>
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</ul>
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<center>
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<table border="0">
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<tr>
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<td> Well contents:</td>
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<td></td>
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</tr>
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<tr>
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<td>Samples</td>
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<td>Contents</td>
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</tr>
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<tr>
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<td>100 µL of PicoGreen in 1x TE buffer</td>
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<td>100 µL of PicoGreen in 1x TE buffer</td>
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</tr>
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<tr>
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<td>99 µL of 1x TE buffer</td>
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<td>100 µL of 1x TE buffer</td>
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</tr>
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<tr>
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<td>1 µL of DNA</td>
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<td> </td>
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</tr>
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</table>
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</center>
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<h2>Calculations</h2>
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<p><i>Note: values less than 1 µL are rounded up.</i></p>
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<ol>
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<li>Volume of PicoGreen and TE buffer needed for the samples</li>
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<ul><li>Volume of 1x TE needed = 100 µL * n</li>
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<li>ie. <i>100 µL x (18 samples + 2 blanks + 1 pipetting) = 2100 µL</i></li>
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<li>n = # of samples + 2 blanks + 1 for pipetting error</li></ul>
 +
 +
<li>Total volume of 1x TE buffer needed</li>
 +
<ul>
 +
<li>Volume of 1x TE needed = (99 µL * n) + volume from step 1</li>
 +
<li>ie. <i>99 µL * (18 samples + 2 blanks + 1 pipetting) + 2100 µL
 +
= 4179 µL</i></li>
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</ul>
 +
 +
<li>Dilution of 20x TE buffer to 1x TE buffer (C1V1=C2V2)</li>
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<ul><li>Volume of 1x TE</li>
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<ul><li>(1/20 dilution * total 1x TE volume) / 1/1 dilution TE</li></li>
 +
<li>(ie. <i>1/20 * 4179 µL = 209 µL of 20x TE buffer</i></li></ul>
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<li>Volume of DNase free water</li>
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<ul><li>Total 1x TE volume – volume of 1x TE</li>
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<li><i>4179 µL – 209 µL = 3970 µL DNase free water</i></li></ul>
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 +
<li>Dilution for PicoGreen dye</li>
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<ul><li>(1/200 dilution of PicoGreen * Volume of PicoGreen) / 1 dilution</li>
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<li>ie. <i>(1/200 dilution of PicoGreen * 2100 µL PicoGreen) / 1 dilution = 10.5 µL of stock PicoGreen</i></li></ul>
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</ol>
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<h2>Procedure</h2>
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<ol>
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<li>To prepare the total volume of 1x TE buffer needed, pipette the volume of 20x TE buffer and DNase free water required into a 15 mL falcon tube. Vortex the solution well to mix. </li>
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<li>Pipette the volume of 1x TE buffer needed to make the PicoGreen mixture into a separate 15mL falcon tube wrapped in aluminum foil and label it quantit. </li>
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<li>Into the aliquot tube pipette the volume of PicoGreen stock needed. Vortex the tube and set aside. </li>
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 +
<li>Prepare the black 96-well microtiter plate by determining where each duplicate of the DNA samples and the two blank wells will be placed on the plate. </li>
 +
 +
<li>Turn on the plate reader. </li>
 +
 +
<li>Pipette 100 µL of 1x TE buffer into each of the sample and blank wells on the black microtiter plate. </li>
 +
 +
<li>Pipette 1µL of each DNA sample into the corresponding well defined by step 4.  Each DNA sample should be run in duplicate to ensure the accuracy of resulting data. </li>
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 +
<li>Pour the 1x TE buffer and PicoGreen quantit into a reservoir and then pipette 99 µL into each well using a multi-channel pipette.  Start a timer after adding the PicoGreen to the first set of wells. Cover the plate with aluminum foil to protect the PicoGreen from light. </li>
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<li>Setup the plate reader software indicating where the sample and blank wells are on the plate. </li>
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<li>After 5 minutes place the plate in the plate reader and click the “Read” button in the menu bar. </li>
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<li>Print the results ensuring that only page 1 of 1 is printing. </li>
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</ol>
</html>
</html>
}}
}}

Latest revision as of 02:53, 4 October 2012

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PicoGreen Assay Protocol – 1/100 Assay

This protocol describes a method of quantifying the amount of DNA in a given sample by fluorescence of the PicoGreen dye when it intercalates in the DNA.

Materials

  • Plate reader
  • Black 96-well plate
  • 15mL Falcon tubes
  • Aluminum foil
  • Ice bucket and ice for DNA samples
  • PicoGreen dye
  • 20x TE buffer
  • DNase free water
  • DNA samples
Well contents:
Samples Contents
100 µL of PicoGreen in 1x TE buffer 100 µL of PicoGreen in 1x TE buffer
99 µL of 1x TE buffer 100 µL of 1x TE buffer
1 µL of DNA

Calculations

Note: values less than 1 µL are rounded up.

  1. Volume of PicoGreen and TE buffer needed for the samples
    • Volume of 1x TE needed = 100 µL * n
    • ie. 100 µL x (18 samples + 2 blanks + 1 pipetting) = 2100 µL
    • n = # of samples + 2 blanks + 1 for pipetting error
  2. Total volume of 1x TE buffer needed
    • Volume of 1x TE needed = (99 µL * n) + volume from step 1
    • ie. 99 µL * (18 samples + 2 blanks + 1 pipetting) + 2100 µL = 4179 µL
  3. Dilution of 20x TE buffer to 1x TE buffer (C1V1=C2V2)
    • Volume of 1x TE
      • (1/20 dilution * total 1x TE volume) / 1/1 dilution TE
      • (ie. 1/20 * 4179 µL = 209 µL of 20x TE buffer
    • Volume of DNase free water
      • Total 1x TE volume – volume of 1x TE
      • 4179 µL – 209 µL = 3970 µL DNase free water
    • Dilution for PicoGreen dye
      • (1/200 dilution of PicoGreen * Volume of PicoGreen) / 1 dilution
      • ie. (1/200 dilution of PicoGreen * 2100 µL PicoGreen) / 1 dilution = 10.5 µL of stock PicoGreen

Procedure

  1. To prepare the total volume of 1x TE buffer needed, pipette the volume of 20x TE buffer and DNase free water required into a 15 mL falcon tube. Vortex the solution well to mix.
  2. Pipette the volume of 1x TE buffer needed to make the PicoGreen mixture into a separate 15mL falcon tube wrapped in aluminum foil and label it quantit.
  3. Into the aliquot tube pipette the volume of PicoGreen stock needed. Vortex the tube and set aside.
  4. Prepare the black 96-well microtiter plate by determining where each duplicate of the DNA samples and the two blank wells will be placed on the plate.
  5. Turn on the plate reader.
  6. Pipette 100 µL of 1x TE buffer into each of the sample and blank wells on the black microtiter plate.
  7. Pipette 1µL of each DNA sample into the corresponding well defined by step 4. Each DNA sample should be run in duplicate to ensure the accuracy of resulting data.
  8. Pour the 1x TE buffer and PicoGreen quantit into a reservoir and then pipette 99 µL into each well using a multi-channel pipette. Start a timer after adding the PicoGreen to the first set of wells. Cover the plate with aluminum foil to protect the PicoGreen from light.
  9. Setup the plate reader software indicating where the sample and blank wells are on the plate.
  10. After 5 minutes place the plate in the plate reader and click the “Read” button in the menu bar.
  11. Print the results ensuring that only page 1 of 1 is printing.

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