Team:Washington/HEAT SHOCK CHEM TRANS

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<b>Heat shock protocol (approximate time 1hrs)</b>
<b>Heat shock protocol (approximate time 1hrs)</b>
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<li>Remove and <b>thaw</b> (on ice) chemically competent cells, either 1.5ml tube or PCR tube.
<li>Remove and <b>thaw</b> (on ice) chemically competent cells, either 1.5ml tube or PCR tube.
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<li>Add 200uL2-1mL of LB and shake for 30 min at 37°C (for higher transformation efficiency, do 1 hr). This is called <b>recovery.</b></li>
<li>Add 200uL2-1mL of LB and shake for 30 min at 37°C (for higher transformation efficiency, do 1 hr). This is called <b>recovery.</b></li>
<li><b>Plate</b> 200uL cells on appropriate antibiotic plate1 and grow O/N in 37°C incubator</li>
<li><b>Plate</b> 200uL cells on appropriate antibiotic plate1 and grow O/N in 37°C incubator</li>
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<b>Tips</b>
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<li>Preheat plates in 37C before for better results (labeling and cell absorption).</li>
<li>Preheat plates in 37C before for better results (labeling and cell absorption).</li>
<li>If using 300ul pcr tubes, omit shaking, incubate 37C.</li>
<li>If using 300ul pcr tubes, omit shaking, incubate 37C.</li>

Latest revision as of 04:34, 27 September 2013


Heat shock protocol (approximate time 1hrs)

  1. Remove and thaw (on ice) chemically competent cells, either 1.5ml tube or PCR tube.
    1. 1.5mL tube aliquots of cells contain approx. 205uL cells
    2. Pcr tubes aliquots contain 50uls
  2. Add 1-5µl DNA to 40-50µl of competent cells.
  3. ncubate on ice for 5 minutes (for higher transformation efficiency, do 30 min)
  4. Heat shock cells for 45 seconds at 42°C
  5. Let cells stand on ice for 2 minutes (or longer)
  6. Add 200uL2-1mL of LB and shake for 30 min at 37°C (for higher transformation efficiency, do 1 hr). This is called recovery.
  7. Plate 200uL cells on appropriate antibiotic plate1 and grow O/N in 37°C incubator

Tips
  1. Preheat plates in 37C before for better results (labeling and cell absorption).
  2. If using 300ul pcr tubes, omit shaking, incubate 37C.

NOTE: Do not use XL10 Gold Cells with Chlor Plates

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