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Materials
You will need:
- Fermentas GeneJET Plasmid Miniprep Kit
- Ethanol (for first use)
- Microcentrifuge tubes
Extra Notes
Buffer preparation
- Add provided RNase A solution to the resuspension solution. Afterwards, it can be used for 6 months, but must be stored at 4°C.
- Add 96%-100% ethanol to wash solution prior to first use.
Purification
- All purification steps are to be done at room temperature
- All centrifugations should be carried out in a table-top microcentrifuge at >12000x g (10,000-14,000 rpm depending on rotor type.)
For more (and detailed) notes and protocol, see pg. 3 in Fermentas protocol booklet
Procedure
Extracting DNA from cells
- Take out previously cultured cells
- Separate cells from luria broth by centrifuging at 4000 rpm for 10 minutes
- Empty out the luria broth completely.
- Add 250μL of the resuspension solution, vortex, then transfer cell suspension to a microcentrifuge tube.
- Add 250μL lysis solution and invert the tube 4-6.
- Add 350μL neutralization solution and mix immediately and thoroughly by inverting the tube 4-6 times.
- Centrifuge for 5 minutes.
Binding DNA
- Transfer supernatant to spin column.
- Centrifuge for 1 minute.
- Discard flowthrough
Washing Column of Residues
- Add 500μL wash solution and centrifuge for 30~60 seconds. Discard flowthrough, place column back into same collection tube.
- Repeat above step with another 500μL wash solution
- Centrifuge empty column 1 minute to remove residual wash solution.
Eluting DNA
- Transfer spin column to fresh 1.5mL microcentrifuge tube.
- Add 50μL elution buffer to center of spin column. Incubate for 2 minutes at room temperature.
- Centrifuge for 2 minutes.
- Keep the purified plasmid DNA at -20°C
Purpose
To purify the plasmid DNA from the cells.
References
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We would like to take a moment to thank all of our sponsors for their very generous donations, as we could not have done this without your help!
We would also like to thank and acknowledge:
Our Advisors
Marc Facciotti
Ilias Tagkopoulos
Technical Guidance
David Larsen
Andrew Yao
Visiting iGEMer
Jia Li of Zhejiang University (TEAM ZJU-China)
cI Promoter Screen
Drew Endy - Stanford
Thomas Schneider - NIH
Want to sponsor us? Send an email to mtfacciotti@ucdavis.edu to discuss various ways you can help! :)
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