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Team:Rice University/Electroporation
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(New page: Electroporation For more information visit www.bio-rad.com and search "electroprotocols manual" ------------------------------------------------------------------------------------------...)
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Revision as of 18:47, 18 June 2008
Electroporation
For more information visit www.bio-rad.com and search "electroprotocols manual"
WHAT YOU WILL NEED: ice bucket electrocompetent cells - located in 1.5mL microcentrifuge tubes in -80*C freezer. Thaw in ice bucket (1 prep per transformation) DNA - purified containing NO-SALT SOC media - recipe located in media folder. Prewarm to 42*C. (1mL per transformation) sterile electroporation cuvettes - located above thermal cycler in cabinet. Chill in ice bucket (1 cuvette per transformation) LB-agar antibiotic plate - located in 4*C far left. Make sure to get plate with appropriate antibiotic. Dry plate out in 37*C incubator at least 30 minutes before plating.
1. Thaw the cells on ice (approx. 40uL).
2. Pippette DNA into tube containing cells and mix gently. Usually 1-2uL of miniprepped DNA will be sufficient. If using less concentrated DNA (i.e. ligation product) up to 10 of DNA can be used.
3. Let DNA and cells incubate on ice for at least 1 min.
4. Turn on micropulser (switch is in the back on the right). And set to Ec1 by pressing the settings button until Ec1 is displayed.
5. Transfer the cells (approx 40uL) into the chilled electroporation cuvette, tapping the cuvette so that the cells make contact with both electrodes and there are no bubbles in path.
6. Place the cuvette into the plastic chamber slide (only fits one way) and push the slide into the chamber until the cuvette is seated between the contacts at the base of the chamber.
7. Pulse once by pressing the pulse button. QUICKLY check the voltage and pulse time by pressing the measurements button. If the voltage is approximately 1.8 kV and the pulse time is approximately ms, the pulse was good. If the time showed as arc, that means there was salt in the prep, or the electroporation cuvette was bad, and the electroporation protocol needs to be redone.
8. QUICKLY resuspend the cells in 1.0 mL of prewarmed 42*C SOC media and place back into 1.5mL centrifuge tube. Incubate for 1 hour @ 37*C, shaking at at 250 rpm.
9. Plate in appropriate dilutions (in sterile 50% gylcerol) on LB-agar antibiotic plate using sterile glass beads. Goal is to obtain isolated colonies. If efficiency of transformation is unsure, plate multiple dilutions. Suggested dilutions 5ul of 1/100,1/1,000, 1/10,000.
