Transforming chemically competent cells (Inoue) protocol

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<html> <h2>Solutions/reagents:</h2><ul type="circle"><li>TB buffer cells</li><li>DNA</li><li>SOC stored at room temperature</li><li>plate made with appropriate antibiotic</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Sterile 1.5-ml microcentrifuge tubes</li></ul><h2>Steps:</h2><ol><li>Measure out 25 - 200 µl of TB buffer cells into sterile 1.5-ml microcentrifuge tube (1). Allow TB buffer cells to thaw on ice. Do not use glass tubes which adsorb DNA. </li><li>Measure out DNA into sterile 1.5-ml microcentrifuge tube (1). Mix solution by pipetting up and down several times. Keep volume of DNA less than 5% of the cell volume. </li><li>Incubate on ice for 30 mins. Note: If you are in a rush, you can shorten this incubation time to 5-10 min. </li><li>Incubate at 42°C for 30 secs. </li><li>Incubate on ice for 2 mins. </li><li>Add 4 volumes SOC. (not critical) </li><li>Incubate at 37°C for 1 hr with shaking at 200 rpm. Note: Can also save some time here by reducing incubation to ~45 min. Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics. </li><li>Plate out 100 - 300 µl of DNA onto plate made with appropriate antibiotic. </li><li>Incubate at 37°C for 12 hrs(overnight). </li></ol></ul></ul>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 13 hrs, 32 mins </html>

Transforming chemically competent cells (Inoue) protocol

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