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Materials
You will need:
- Fermentas GeneJET Gel Extraction Kit
- Microcentrifuge tubes
- Microtiter pipette & tips
Extra Notes
- All steps should be carried out at room temperature.
- All centrifugations should be carried out in a microcentrifuge at > 12,000xg (10,000-14,000 rpm, depending on the rotor type)
- See Fermentas protocol booklet for more information
Procedure
- Cut out gel slice with the desired DNA fragment and weigh gel.
- Add 1:1 volume of binding buffer to gel slice. (Eg. 100μL of binding buffer for every 100mg of agarose gel.)
- Incubate gel solution at 50°C-60°C for 10 minutes. Inverting tube helps melting process.
- (Optional: only use if DNA fragment is <500bp or >10kb long) If DNA fragment is <500bp, add a 1:2 volume of 100% isopropanol to the solubilized gel solution. Mix thoroughly. If DNA fragment is >10kb, add a 1:2 volume of water to the solubilized gel solution. Mix thoroughly.
- Transfer (up to 800μL) gel solution to spin column.
- Centrifuge for 30-60 seconds. Discard flow-through and place spin column back into same collection tube.
- Add 700μL of wash buffer and centrifuge for 1 minute.
- Discard flow-through and the centrifuge empty column for 1 minute.
- Place the column into a fresh 1.5mL microcentrifuge tube.
- Add 50μL of elution buffer to the column.
- Centrifuge for 1 minute. Keep the flow-through.
Purpose
To purify the DNA from the gel.
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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