Team:Macquarie Australia/WetLab/Protocols/PlasmidPreps

From 2014.igem.org

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<h3>Plasmid Prep</h3>
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<h3>Plasmid Prep</h3><br/>
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<li>Centrifuge @13,200 rpm for 10 min of 1.8mL of overnight cultures in 2mL Eppendorf’s</li>
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<li>Discard supernatant and add 1.9 mL of culture and centrifuge again @13,200 rpm for 10 min</li>
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<li>Re-suspend pelleted cells in P1 Buffer (250µl) </li>
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<li>Add 250µL of P2 buffer & invert 4-6 times (turns homogenous blue)</li>
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<li>Add 350µL of N3 & mix by inverting (turns colourless)</li>
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<li>Centrifuge @ 10min 13,000 rpm to obtain a pellet</li>
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<li>Transfer supernatant in QIA Prep Spin Column by pipetting</li>
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<li>Centrifuge 30-60 sec - discard flow through</li>
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<li>Wash QIA Prep Spin Column with 0.5mL of PB</li>
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<li>Centrifuge for 30-60 seconds - discard flow through</li>
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<li>Wash spin column by adding 0.75 mL PE buffer</li>
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<li>Centrifuge for 30-60 seconds - discard flow through and centrifuge for a further 1min</li>
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<li>Place QIAPrep Column in clean Eppendorf</li>
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<li>Elute DNA add 50µl of water, stand for 1min, centrifuge for 1min</li>
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<li>QIA prep - Spin miniprep buffer</li>
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Revision as of 05:09, 16 October 2014

Protocols


Plasmid Prep


  • Centrifuge @13,200 rpm for 10 min of 1.8mL of overnight cultures in 2mL Eppendorf’s
  • Discard supernatant and add 1.9 mL of culture and centrifuge again @13,200 rpm for 10 min
  • Re-suspend pelleted cells in P1 Buffer (250µl)
  • Add 250µL of P2 buffer & invert 4-6 times (turns homogenous blue)
  • Add 350µL of N3 & mix by inverting (turns colourless)
  • Centrifuge @ 10min 13,000 rpm to obtain a pellet
  • Transfer supernatant in QIA Prep Spin Column by pipetting
  • Centrifuge 30-60 sec - discard flow through
  • Wash QIA Prep Spin Column with 0.5mL of PB
  • Centrifuge for 30-60 seconds - discard flow through
  • Wash spin column by adding 0.75 mL PE buffer
  • Centrifuge for 30-60 seconds - discard flow through and centrifuge for a further 1min
  • Place QIAPrep Column in clean Eppendorf
  • Elute DNA add 50µl of water, stand for 1min, centrifuge for 1min
  • QIA prep - Spin miniprep buffer

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