Transforming chemically competent cells protocol

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Revision as of 23:59, 22 October 2009 by Vaishnavi Ananth (talk | contribs) (New page: <html> <h2>Solutions/reagents:</h2><ul type="circle"><li>TSS cells</li><li>DNA</li><li>SOC stored at room temperature</li><li>plate made with appropriate antibiotic</li></ul><h2>Equipment:...)
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<html> <h2>Solutions/reagents:</h2><ul type="circle"><li>TSS 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><p><li>Measure out TSS cells into sterile 1.5-ml microcentrifuge tube (1).<br>Allow TSS cells to thaw on <b><font color=#357EC7>ice</font></b>.<br></li></p><p><li>Add <font color=#357EC7>DNA</font> to TSS cells.<br><font color = "#800517"><i>(1µl of prepped plasmid is more than enough)</i></font><br>Mix solution by pipetting up and down several times.<br><font color = "#800517"><i>Note: If you are adding small volumes (~1µl), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.</i></font><br></li></p><p><li>Store the tube <b><font color=#357EC7>on ice</font></b> for <b><font color=#357EC7>30 mins</font></b>.<br><font color = "#800517"><i>Note: If you are in a rush, you can shorten this incubation time to 5-10 min.</i></font><br></li></p><p><li>Incubate at <b><font color=#357EC7>42°C</font></b> for <b><font color=#357EC7>30 secs</font></b>.<br><font color = "#800517"><i>Note: According to the original TSS paper and qualitative experience (JM), this step is completely optional and may actually reduce transformation efficiency.</i></font><br></li></p><p><li>Incubate on <b><font color=#357EC7><b><font color=#357EC7>ice</font></b></font></b> for <b><font color=#357EC7>2 mins</font></b>.<br></li></p><p><li>Add <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>SOC</font>.<br><font color = "#800517"><i>(2XYT and LB are also suitable, original paper suggests LB + 20mM glucose)</i></font><br></li></p><p><li>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>1 hr</font></b> with shaking at 200 rpm.<br><font color = "#800517"><i>Note: Can also save some time here by reducing incubation to ~45 min.</i></font><br></li></p><p><li>Plate out <b><font color=#357EC7>100 - 300 µl</font></b> of SOC onto <font color=#357EC7>plate made with appropriate antibiotic</font>.<br></li></p><p><li>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>12 hrs</font></b>(overnight).<br></li></p><p><li><font color = "#800517"><i>Save the rest of the transformants in liquid culture at 4°C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.</i></font><br></li></p></ol> </html>