Transforming chemically competent cells (Inoue) protocol

Solutions/reagents: TB buffer cellsDNASOC stored at room temperatureplate made with appropriate antibiotic Equipment: IncubatorSterile 1.5-ml microcentrifuge tubes Steps:  Measure out <font color=#357EC7>25 - 200 µl  of <font color=#357EC7>TB buffer cells into sterile 1.5-ml microcentrifuge tube (1). Allow TB buffer cells to thaw on <font color=#357EC7>ice . <font color = "#800517">Do not use glass tubes which adsorb DNA. </li> Measure out DNA into sterile 1.5-ml microcentrifuge tube (1). Mix solution by pipetting up and down several times. <font color = "#800517">Keep volume of DNA less than 5% of the cell volume. </li> Incubate on <font color=#357EC7><b><font color=#357EC7>ice  </b> for <font color=#357EC7>30 mins . <font color = "#800517">Note: If you are in a rush, you can shorten this incubation time to 5-10 min. </li> Incubate at <font color=#357EC7>42°C  for <font color=#357EC7>30 secs . </li> Incubate on <font color=#357EC7><b><font color=#357EC7>ice  </b> for <font color=#357EC7>2 mins . </li> Add  <font color=#357EC7>4  volumes <font color=#357EC7>SOC. <font color = "#800517">(not critical) </li> Incubate at <font color=#357EC7>37°C  for <font color=#357EC7>1 hr  with shaking at 200 rpm. <font color = "#800517">Note: Can also save some time here by reducing incubation to ~45 min. <font color = "#800517">Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics. </li> Plate out <font color=#357EC7>100 - 300 µl  of DNA onto <font color=#357EC7>plate made with appropriate antibiotic. </li> Incubate at <font color=#357EC7>37°C  for <font color=#357EC7>12 hrs (overnight). </li> </ol></ul></ul> TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 13 hrs, 32 mins