Transforming chemically competent cells protocol

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(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:...)
Current revision (01:45, 20 November 2009) (view source)
 
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<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>
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<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></ul><h2>Steps:</h2><ol><p><li>Allow TSS cells to thaw on <b><font color=#357EC7>ice</font></b>.<br></li></p><p><li>Measure out DNA into sterile 1.5-ml microcentrifuge tube (1).<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><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 13 hrs, 32 mins</font></b></p>
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Current revision

Solutions/reagents:

  • TSS cells
  • DNA
  • SOC stored at room temperature
  • plate made with appropriate antibiotic

Equipment:

  • Incubator

Steps:

  1. Allow TSS cells to thaw on ice.
  2. Measure out DNA into sterile 1.5-ml microcentrifuge tube (1).
    (1µl of prepped plasmid is more than enough)
    Mix solution by pipetting up and down several times.
    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.
  3. Store the tube on ice for 30 mins.
    Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
  4. Incubate at 42°C for 30 secs.
    Note: According to the original TSS paper and qualitative experience (JM), this step is completely optional and may actually reduce transformation efficiency.
  5. Incubate on ice for 2 mins.
  6. Add 1 ml of SOC.
    (2XYT and LB are also suitable, original paper suggests LB + 20mM glucose)
  7. 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.
  8. Plate out 100 - 300 µl of SOC onto plate made with appropriate antibiotic.
  9. Incubate at 37°C for 12 hrs(overnight).
  10. 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.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 13 hrs, 32 mins

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