Team:Paris/Miniprep

From 2009.igem.org

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(→Adaptation of PureYield(TM) Plasmid Miniprep System (Promega))
(→Prepare Lysate)
 
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==Adaptation of PureYield&tade; Plasmid Miniprep System (Promega)==
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==Adaptation of PureYield™ Plasmid Miniprep System (Promega)==
===Prepare Lysate===
===Prepare Lysate===
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*Put 2ml of bacterial culture in a 2ml microcentrifuge tube.
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#Put 2ml of bacterial culture in a 2ml microcentrifuge tube.
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*Centrifuge for 30 seconds at maximum speed in a microcentrifuge. Discard the supernatant.
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#Centrifuge for 30 seconds at maximum speed in a microcentrifuge. Discard the supernatant.
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*Add 600µl of H<sub>2</sub>O (Gibco), and resuspend completely.
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#Add 600µl of H<sub>2</sub>O (Gibco), and resuspend completely.
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*Add 100µl of Cell Lysis Buffer (Blue), and mix by inverting the tube 6 times.
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#Add 100µl of Cell Lysis Buffer (Blue), and mix by inverting the tube 6 times.
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*Wait 2min but no more than 5min !
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#Wait 2min but no more than 5min !
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*Add 350µl of cold (4-8°C) Neutralization Solution and immediatly mix thoroughly by inverting.
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#Add 350µl of cold (4-8°C) Neutralization Solution and immediatly mix thoroughly by inverting.
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*Centrifuge at maximum speed in a microcentrifuge for 10 minutes.
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#Centrifuge at maximum speed in a microcentrifuge for 10 minutes.
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*Place a PureYield(TM) minicolumn on a Luer-Lok(R) adapter of a VacMan(R).
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#Place a PureYield&trade; minicolumn on a Luer-Lok&reg; adapter of a VacMan(R).
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*Transfer the supernatant (~900µl) into the PureYield(TM) minicolumn without disturbing the cell debris pellet (yellow-orange).
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#Transfer the supernatant (~900µl) into the PureYield&trade; minicolumn without disturbing the cell debris pellet (yellow-orange).
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*Apply vacuum pulling the lysate through the column.
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#Apply vacuum pulling the lysate through the column.
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<br>
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===Wash===
===Wash===
*Add 200µl of Endotoxin Removal Wash (ERB) to the minicolumn. Allow the vacuum to pull the solution through the column.
*Add 200µl of Endotoxin Removal Wash (ERB) to the minicolumn. Allow the vacuum to pull the solution through the column.
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*Add 400µl of Column Wash Solution (CWC) to the minicolumn. Allow the vacuum to pull the solution through the column. Release the vacuum, and remove the PureYield(TM) Minicolumn.
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*Add 400µl of Column Wash Solution (CWC) to the minicolumn. Allow the vacuum to pull the solution through the column. Make sure there is no more ethanol droplets in the column. Release the vacuum, and remove the PureYield&trade; Minicolumn.
*Let dry for 5 minutes. (Preheat Nuclease Free Water at 37°C).
*Let dry for 5 minutes. (Preheat Nuclease Free Water at 37°C).
===Elute===
===Elute===
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*Place the column in a 2ml collection tube, and centrifuge at maximum speed in a microcentrifuge for 2 minutes.
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*Place the column in a 2ml collection tube (from the kit), and centrifuge at maximum speed in a microcentrifuge for 2 minutes, twice.
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*Transfer the minicolumn into a clean 1.5ml microcentrifuge tube, then add 50µl of hot nuclease-free water directly to the minicolumn matrix. Let stand for 1 minute at room temperature.
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*Transfer the minicolumn into a clean 1.5ml Eppendorf tube, then add 52µl of 37°C nuclease-free water (Gibco) directly to the minicolumn matrix. Let stand for 1 minute at room temperature.
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*Centrifuge for 1 minute to eluate the plasmid DNA. Cap the microcentrifuge tube, and store eluted plasmid DNA at -20°C.
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*Centrifuge for 1 minute to eluate the plasmid DNA. Cap the microcentrifuge tube, label it and store the eluted plasmid DNA at -20°C.

Latest revision as of 12:56, 10 August 2009

Contents

Adaptation of PureYield™ Plasmid Miniprep System (Promega)

Prepare Lysate

  1. Put 2ml of bacterial culture in a 2ml microcentrifuge tube.
  2. Centrifuge for 30 seconds at maximum speed in a microcentrifuge. Discard the supernatant.
  3. Add 600µl of H2O (Gibco), and resuspend completely.
  4. Add 100µl of Cell Lysis Buffer (Blue), and mix by inverting the tube 6 times.
  5. Wait 2min but no more than 5min !
  6. Add 350µl of cold (4-8°C) Neutralization Solution and immediatly mix thoroughly by inverting.
  7. Centrifuge at maximum speed in a microcentrifuge for 10 minutes.
  8. Place a PureYield™ minicolumn on a Luer-Lok® adapter of a VacMan(R).
  9. Transfer the supernatant (~900µl) into the PureYield™ minicolumn without disturbing the cell debris pellet (yellow-orange).
  10. Apply vacuum pulling the lysate through the column.

Wash

  • Add 200µl of Endotoxin Removal Wash (ERB) to the minicolumn. Allow the vacuum to pull the solution through the column.
  • Add 400µl of Column Wash Solution (CWC) to the minicolumn. Allow the vacuum to pull the solution through the column. Make sure there is no more ethanol droplets in the column. Release the vacuum, and remove the PureYield™ Minicolumn.
  • Let dry for 5 minutes. (Preheat Nuclease Free Water at 37°C).

Elute

  • Place the column in a 2ml collection tube (from the kit), and centrifuge at maximum speed in a microcentrifuge for 2 minutes, twice.
  • Transfer the minicolumn into a clean 1.5ml Eppendorf tube, then add 52µl of 37°C nuclease-free water (Gibco) directly to the minicolumn matrix. Let stand for 1 minute at room temperature.
  • Centrifuge for 1 minute to eluate the plasmid DNA. Cap the microcentrifuge tube, label it and store the eluted plasmid DNA at -20°C.

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