Preparing chemically competent cells

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*Grow a 5-25mL culture to an OD<sub>600</sub> of 0.2 <math>\rightarrow</math> 0.5
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==Making==
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*Centrifuge for 10 minutes at 3000rpm and <math>4^o</math>C.
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#Grow a 5 - 25 mL culture to an OD<sub>600</sub> of 0.2 - 0.5.
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*Remove supernatant and replace with 10% original volume [[TSS]].
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#Centrifuge for 10 minutes at 3000 rpm and <math>4^o</math>C.
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*Make 100uL aliquots and store at <math>-80^o</math>C.
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#Remove supernatant and replace with 10% original volume [[TSS]].
 +
#Make 100 uL aliquots and store at <math>-80^o</math>C.
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==Using==
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#Thaw TSS cells on ice.
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#Add DNA, pipette gently to mix.
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#Let sit 30 minutes on ice.
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#Incubate cells for 30 seconds at <math>42^o</math>C.
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#Add 1 mL [[SOC]] (Room Temp).
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#Incubate for 1 hour at <math>37^o</math>C.
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#Plate 200 µL onto appropriate resistance plate.

Revision as of 15:24, 6 June 2005

Making

  1. Grow a 5 - 25 mL culture to an OD600 of 0.2 - 0.5.
  2. Centrifuge for 10 minutes at 3000 rpm and 4oC.
  3. Remove supernatant and replace with 10% original volume TSS.
  4. Make 100 uL aliquots and store at − 80oC.

Using

  1. Thaw TSS cells on ice.
  2. Add DNA, pipette gently to mix.
  3. Let sit 30 minutes on ice.
  4. Incubate cells for 30 seconds at 42oC.
  5. Add 1 mL SOC (Room Temp).
  6. Incubate for 1 hour at 37oC.
  7. Plate 200 µL onto appropriate resistance plate.


TSS (50 mL)

  • 5g PEG 8000
  • 1.5 mL 1M MgCl2
  • 2.5 mL DMSO
  • LB to 50 mL
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