Transforming chemically competent cells (Inoue) protocol

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(New page: <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>Equi...)
Current revision (02:17, 20 November 2009) (view source)
 
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<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><p><li>Measure out <b><font color=#357EC7>25 - 200 µl</font></b> of <font color=#357EC7>TB buffer cells</font> into sterile 1.5-ml microcentrifuge tube (1).<br>Allow TB buffer cells to thaw on <b><font color=#357EC7>ice</font></b>.<br><font color = "#800517"><i>Do not use glass tubes which adsorb DNA.</i></font><br></li></p><p><li>Add <font color=#357EC7>DNA</font> to TB buffer cells.<br>Mix solution by pipetting up and down several times.<br><font color = "#800517"><i>Keep volume of DNA less than 5% of the cell volume.</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>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></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>4</font></b> volumes <font color=#357EC7>SOC</font>.<br><font color = "#800517"><i>(not critical)</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><font color = "#800517"><i>Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics.</i></font><br></li></p><p><li>Plate out <b><font color=#357EC7>100 - 300 µl</font></b> of DNA 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></ol>
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<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><p><li>Measure out <b><font color=#357EC7>25 - 200 µl</font></b> of <font color=#357EC7>TB buffer cells</font> into sterile 1.5-ml microcentrifuge tube (1).<br>Allow TB buffer cells to thaw on <b><font color=#357EC7>ice</font></b>.<br><font color = "#800517"><i>Do not use glass tubes which adsorb DNA.</i></font><br></li></p><p><li>Measure out DNA into sterile 1.5-ml microcentrifuge tube (1).<br>Mix solution by pipetting up and down several times.<br><font color = "#800517"><i>Keep volume of DNA less than 5% of the cell volume.</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>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></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>4</font></b> volumes <font color=#357EC7>SOC</font>.<br><font color = "#800517"><i>(not critical)</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><font color = "#800517"><i>Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics.</i></font><br></li></p><p><li>Plate out <b><font color=#357EC7>100 - 300 µl</font></b> of DNA 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></ol></ul></ul><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:

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

Equipment:

  • Incubator
  • Sterile 1.5-ml microcentrifuge tubes

Steps:

  1. 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.
  2. 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.
  3. Incubate 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.
  5. Incubate on ice for 2 mins.
  6. Add 4 volumes SOC.
    (not critical)
  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.
    Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics.
  8. Plate out 100 - 300 µl of DNA onto plate made with appropriate antibiotic.
  9. Incubate at 37°C for 12 hrs(overnight).

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

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