Transforming chemically competent cells (Inoue)

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(Method)
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#Spread 100-300 μl onto a plate made with appropriate antibiotic.
#Spread 100-300 μl onto a plate made with appropriate antibiotic.
#Grow overnight at 37°C.
#Grow overnight at 37°C.
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==Experimental results==
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First attempt varied several parameters: incubation time on ice prior to heat shock, heat shock length, addition of DTT at 20mM.
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* DTT appeared to have little effect when added during transformation.
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* Incubating for 1/2 hour on ice had a positive effect, perhaps 1.5 to 2x efficiency gain.
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* Heat shock of 0 or 15 s rather than 30 s reduced efficiency about 8x
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* Heat shock at 30 s or 60 s gave approximately similar results.
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Achieved efficiency was 3 x 10<sup>7</sup> per microgram.  A control transformation with Invitrogen cells was at 1.2 x 10<sup>8</sup> per microgram.
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[[Category:E.Coli Protocol]]
[[Category:E.Coli Protocol]]

Revision as of 13:41, 18 April 2006

Also see Preparing chemically competent cells (Inoue)

Method

  1. Thaw 25 - 200 μl TB buffer cells on ice. Do not use glass tubes, which adsorb DNA.
  2. Add DNA, pipette gently to mix (keep volume of DNA less than 5% of the cell volume)
  3. Incubate on ice for 30 minutes
    • Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
  4. Incubate cells for 30 seconds at 42°C.
  5. Incubate cells on ice for 2 min.
  6. Add 4 volumes of room temperature SOC (not critical)
  7. Incubate for 1 hour at 37°C on shaker.
    • 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. Spread 100-300 μl onto a plate made with appropriate antibiotic.
  9. Grow overnight at 37°C.

Experimental results

First attempt varied several parameters: incubation time on ice prior to heat shock, heat shock length, addition of DTT at 20mM.

  • DTT appeared to have little effect when added during transformation.
  • Incubating for 1/2 hour on ice had a positive effect, perhaps 1.5 to 2x efficiency gain.
  • Heat shock of 0 or 15 s rather than 30 s reduced efficiency about 8x
  • Heat shock at 30 s or 60 s gave approximately similar results.

Achieved efficiency was 3 x 107 per microgram. A control transformation with Invitrogen cells was at 1.2 x 108 per microgram.

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