Difference between revisions of "Team:Paris Bettencourt/Protocols"
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</div> | </div> | ||
+ | |||
+ | <div class="textBox" id="ElectrocompetentLactobacillus"> | ||
+ | <h3><i>Lactobacillus plantarum</i> electrocompetent cells</h3> | ||
+ | <b>Electrocompetent Cells Preparation Protocol for Lactobacillus Plantarum</b> | ||
+ | <ul> | ||
+ | |||
+ | <li> Inoculate 5ml MRS medium with L. plantarum freezer stock.</li> | ||
+ | <li> Grow overnight at 30°C without shaking.</li> | ||
+ | <li> Add 1.25g glycine to two flasks of 50ml MRS medium.</li> | ||
+ | <li> Shake to dissolve.</li> | ||
+ | <li> Add 1ml overnight culture to each flask (1:50 dlution).</li> | ||
+ | <li> Culture for ~3 hours at 37°C with shaking.</li> | ||
+ | <li> Centrifuge culture for 5min at 4000g</li> | ||
+ | <li> Resuspend in 25ml ice-cold DI water.</li> | ||
+ | <li> Repeat it.</li> | ||
+ | <li> Centrifuge for 5min at 4000g</li> | ||
+ | <li> Resuspend in 5ml 50mM EDTA.</li> | ||
+ | <li> Incubate on ice for 5 minutes</li> | ||
+ | <li> Add 25ml ice-cold DI water.</li> | ||
+ | <li> Centrifuge culture for 5min at 4000g</li> | ||
+ | <li> Resuspend in 25ml ice-cold DI water.</li> | ||
+ | <li> Centrifuge culture for 5min at 4000g</li> | ||
+ | <li> Resuspend in 25ml ice-cold 0.5M Sucrose, 10% glycerol.</li> | ||
+ | <li> Repeat it.</li> | ||
+ | <li> Centrifuge culture for 5min at 4000g</li> | ||
+ | <li> Resuspend in 0.8ml ice-cold 0.5M Sucrose, 10% glycerol.</li> | ||
+ | <li> Aliquot 90μL of cell concentrate into ~20 microcentrifuge tubes.</li> | ||
+ | <li> Keep on ice until use (within the next two hours).</li> | ||
+ | <li> Add 10μL of plasmid DNA to the 90μL of cell concentrate.</li> | ||
+ | <li> Keep on ice for 5 minutes.</li> | ||
+ | <li> Put 1mm cuvettes on ice too.</li> | ||
+ | <li> Pipette the cell/DNA mixture into the cuvette</li> | ||
+ | <li> Electrporate at 1200 volts</li> | ||
+ | <li> Time constant should be ~5.0</li> | ||
+ | <li> Immediately transfer the electroporated cells to 900μL of MRS medium.</li> | ||
+ | <li> Incubate at 30°C for 2-3 hours.</li> | ||
+ | <li> Plate on medium with the appropriate antibiotic.</li> | ||
+ | <li> Incubate at 30°C for two days.</li> | ||
+ | <li> Pick a colony</li> | ||
+ | </ul> | ||
+ | </div> | ||
Revision as of 09:10, 11 September 2015