Team:Amsterdam/project/protocols/transformation dh5 alpha

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<li>Incubate on ice for 30 minutes.</li>
<li>Incubate on ice for 30 minutes.</li>
<li>Heat shock for 45 seconds in a water bath at 42°C, then quickly back on ice for 2 minutes.</li>
<li>Heat shock for 45 seconds in a water bath at 42°C, then quickly back on ice for 2 minutes.</li>
-
<li>Add 450 ml of room temperature SOC medium (Work sterile!!).</li>
+
<li>Add 500 ul of room temperature SOC medium (Work sterile!!).</li>
<li>Incubate 1 hour at 37°C for antibiotic resistance expression, while shaking (225 rpm).</li>
<li>Incubate 1 hour at 37°C for antibiotic resistance expression, while shaking (225 rpm).</li>
<li>Spread 1/10 and 9/10 on LB plates (containing the right antibiotic).</li>
<li>Spread 1/10 and 9/10 on LB plates (containing the right antibiotic).</li>

Revision as of 13:06, 24 July 2012

Transformation Protocol in DH5α (Invitrogen)


  • Thaw an aliquot of competent bacteria on ice.
  • Add 50 µl of DH5α competent cells gently in a sterile 15 ml polypropylene tube. Add 1 µl of ligation mixture or Gibson Assembly reaction (1 – 10 ng of DNA) to the polypropylene tube
  • Incubate on ice for 30 minutes.
  • Heat shock for 45 seconds in a water bath at 42°C, then quickly back on ice for 2 minutes.
  • Add 500 ul of room temperature SOC medium (Work sterile!!).
  • Incubate 1 hour at 37°C for antibiotic resistance expression, while shaking (225 rpm).
  • Spread 1/10 and 9/10 on LB plates (containing the right antibiotic).

    • Notes: Do not shake the polypropylene tubes during the heat shock! To increase the yield, centrifuge gently (at low speed), remove the excess of medium and then spread on the plates. To determine transformation efficiency

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