Team:Macquarie Australia/Protocols and Other Methods

From 2010.igem.org

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<li>Spin at 14,000 rpm for 3 minutes and recover 160uL </li> </ol></p>
<li>Spin at 14,000 rpm for 3 minutes and recover 160uL </li> </ol></p>
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<p> <li type="disc">
See the BioLine manual for full protocol:
See the BioLine manual for full protocol:
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http://www.bioline.com/pdf/guides/Nucleic%20Acid%20Isolation%20Guide.pdf
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http://www.bioline.com/pdf/guides/Nucleic%20Acid%20Isolation%20Guide.pdf </li> </menu>
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<hr><h1>
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Promega Wizard® Plus SV Minipreps DNA Purification System: </h1>
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<ol>
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<li>Centrifuge bacterial culture at 12,000 rpm for 5 minutes, pour off the supernatant and blot the inverted tube on a paper towel to remove excess media</li>
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<li>Add 250uL of Cell Resuspension Solution and completely resuspend the pellet by pippetting</li>
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<li>Add 250uL of Cell Lysis Solution and mix by inverting tube 4 times, incubate for no more than 5 minutes </li>
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<li>Add 10uL Alkaline Protease Solution and mix by inverting tube 4 times, incubate at room temperature for no more than 5 minutes</li>
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<li>Add 350uL of Neutralization Solution and immediately mix by inverting the tube 4 times</li>
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<li>Centrifuge the bacterial lysate at 14,000 rpm in a microcentrifuge for 10 minutes at room temperature</li>
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<li>Transfer cleared lysate to Spin Column by decanting without disturbing precipitate</li>
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<li>Centrifuge the supernatant at 14,000 rpm for 1 minute at room temperature, discard flowthrough</li>
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<li>Add 750uL of Column Wash Solution (diluted with 95% ethanol), to Spin Column</li>
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<li>Centrifuge at 14,000 rpm for 1 minute at room temperature, discard flow through</li>
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<li>Add 250uL of Column Wash Solution</li>
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<li>Centrifuge at 14,000 rpm for 2 minutes at room temperature</li>
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<li>Transfer the Spin Column to a new tube</li>
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<li>Elute the plasmid DNA by adding 100uL of Nuclease Free Water to the Spin Column</li>
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<li>Centrifuge at 14,000 rpm for 1 minute at room temperature</li>
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<li>Add 11uL 10x TE buffer for DNA storage at 4C </li>
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<p> <li type="disc">
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Please note that we made a minor modification to this protocol:
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• 50uL of ssH20 added instead of 100uL for elution step </li>
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<li type="disc"> See the Promega manual for full protocol:
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http://www.promega.com/tbs/tb225/tb225.pdf </li>

Revision as of 09:29, 19 October 2010

Protocols and Other Methods Used

BioLine Isolate Genomic DNA Mini Kit:

  1. Pellet cell culture, decant, add 1mL CLS-TC
  2. Resuspend and transfer to Fastprep tube (BIO101)
  3. Fastprep 5.5g for 30 seconds, then spin @ 14,000 rpm for 5 minutes
  4. Recover 700uL then 700uL Binding Matrix
  5. Rotate for 5 minutes then spin for 1 minute
  6. Decant the supernatant and add 800uL SEWS then vortex
  7. Rotate for 5 minutes then spin for 1 minute
  8. Decant the supernatant then pulse spin and remove the last bit of ethanol
  9. Air dry for 5 minutes then resuspend in 200uL TE buffer
  10. Spin at 14,000 rpm for 3 minutes and recover 160uL

  • See the BioLine manual for full protocol: http://www.bioline.com/pdf/guides/Nucleic%20Acid%20Isolation%20Guide.pdf

    • Promega Wizard® Plus SV Minipreps DNA Purification System:

    1. Centrifuge bacterial culture at 12,000 rpm for 5 minutes, pour off the supernatant and blot the inverted tube on a paper towel to remove excess media
    2. Add 250uL of Cell Resuspension Solution and completely resuspend the pellet by pippetting
    3. Add 250uL of Cell Lysis Solution and mix by inverting tube 4 times, incubate for no more than 5 minutes
    4. Add 10uL Alkaline Protease Solution and mix by inverting tube 4 times, incubate at room temperature for no more than 5 minutes
    5. Add 350uL of Neutralization Solution and immediately mix by inverting the tube 4 times
    6. Centrifuge the bacterial lysate at 14,000 rpm in a microcentrifuge for 10 minutes at room temperature
    7. Transfer cleared lysate to Spin Column by decanting without disturbing precipitate
    8. Centrifuge the supernatant at 14,000 rpm for 1 minute at room temperature, discard flowthrough
    9. Add 750uL of Column Wash Solution (diluted with 95% ethanol), to Spin Column
    10. Centrifuge at 14,000 rpm for 1 minute at room temperature, discard flow through
    11. Add 250uL of Column Wash Solution
    12. Centrifuge at 14,000 rpm for 2 minutes at room temperature
    13. Transfer the Spin Column to a new tube
    14. Elute the plasmid DNA by adding 100uL of Nuclease Free Water to the Spin Column
    15. Centrifuge at 14,000 rpm for 1 minute at room temperature
    16. Add 11uL 10x TE buffer for DNA storage at 4C

    17. Please note that we made a minor modification to this protocol: • 50uL of ssH20 added instead of 100uL for elution step
    18. See the Promega manual for full protocol: http://www.promega.com/tbs/tb225/tb225.pdf

      19. Retrieved from "http://2010.igem.org/Team:Macquarie_Australia/Protocols_and_Other_Methods"