McClean: PlasmidPrep QiagenKit

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Contents

Overview

Please make sure to include:

  • Protocol summarized from the Qiagen kit protocol
  • A link to the nanodrop instructions for quantifying your plasmid yield Using the Nanodrop2000
  • Any information that is McClean Lab specific (where to get water, where the nanodrop is, etc)


Materials

  • RNase A Solution
  • Buffer P1
  • Ethanol (96-100%)
  • Buffer PE
  • Buffer P2
  • Buffer N3

Incubate the bacteria that has your desired plasmid overnight at 37 degrees Celsius

Before completing Plasmid Prep, add both the RNase A solution and Buffer P1 to tube and store at 2-8 degrees Celsius.

Add ethanol and Buffer PE.

At room temp, for 3 mins pellet 1-5mL of bacterial culture in centrifuge at >8000 rpm. Centrifuge for 5-6 minutes at 3700 rpm (our centrifuge does not reach 8000 rpm)

  • COMMENT: 5mL of bacterial culture results in a higher yield. I recommend it. Today's 5mL overnight culture yielded 120ng/uL of DNA -Cameron Stewart, Sept 22nd, 2015

Resuspend pelleted bacterial cells in 250 micro-liters Buffer P1 and transfer to a microcentrifuge tube (eppendorf tube).

Then, add 250 micro-liters of Buffer P2 and mix by inversion until solution becomes clear (4-6 times). Take caution, do not allow reaction to occur >5 mins.

Add 350 micro-liters of Buffer N3 and mix immediately by inversion. Centrifuge mixture for 10 mins at 13000 rpm.

Transfer the supernatant by pipette to a spin column, centrifuge for 30-60s, and discard the flow through.

Add 500 micro-liters of PB Buffer, centrifuge for 30-60s, and discard the flow through.

Add 750 micro-liters of PE Buffer, centrifuge for 30-60s, and discard the flow-through. Centrifuge again for 1 minute to remove leftover flow-through.

Transfer the spin column to a clean microcentrifuge tube. Add 50 micro-liters of EB Buffer, let it stand for 1 minute, and centrifuge for another minute.

Notes

  • Store Buffer P1 at 2-8C
  • Maker sure to immediately mix by inversion when Buffer N3 is added.


References

Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.

Contact

or instead, discuss this protocol.

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