McClean: PlasmidPrep QiagenKit
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)
- 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.
- Store Buffer P1 at 2-8C
- Make sure to immediately mix by inversion when Buffer N3 is added.
- Taylor D. Scott (talk) 11:55, 20 August 2019 (PDT): You can prep in one day if you grow your cultures in Terrific Broth (TB) instead of LB. I've found that 7 hr of growth in TB gives plenty of plasmid for restriction digest or sequencing.
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.
- Megan N McClean 14:01, 20 July 2011 (EDT)
or instead, discuss this protocol.