Team:Washington/Protocols/Transformation

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=General Transformation Protocol=
=General Transformation Protocol=
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#Thaw 20 μl CCMB buffer cells on ice.  Do not use glass tubes, which adsorb DNA.
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#Thaw 20 μl of frozen competent cells on ice.  Do not use glass tubes, which adsorb DNA.
#Add 20 μl DNA, pipette gently to mix (keep volume of DNA less than 5% of the cell volume)
#Add 20 μl DNA, pipette gently to mix (keep volume of DNA less than 5% of the cell volume)
#Incubate on ice for 30 minutes
#Incubate on ice for 30 minutes
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#*Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
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::Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
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#Incubate cells for 30-90 (1 min) seconds at 42°C.
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#Incubate cells for 45-90 seconds at 42°C.
#Incubate cells on ice for 2 min.
#Incubate cells on ice for 2 min.
#Add 200mL of room temperature TB.
#Add 200mL of room temperature TB.
#Incubate for 1 hour at 37°C on shaker.
#Incubate for 1 hour at 37°C on shaker.
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#*Note: Can also save some time here by reducing incubation to ~45 min.
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::Note: Can also save some time here by reducing incubation to ~45 min.
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#*Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics
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::Note: Step can be eliminated if plating on Amp plates, but not other antibiotics
#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.

Latest revision as of 02:42, 16 September 2011


General Transformation Protocol

  1. Thaw 20 μl of frozen competent cells on ice. Do not use glass tubes, which adsorb DNA.
  2. Add 20 μl 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.
  1. Incubate cells for 45-90 seconds at 42°C.
  2. Incubate cells on ice for 2 min.
  3. Add 200mL of room temperature TB.
  4. 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 other antibiotics
  1. Spread 100-300 μl onto a plate made with appropriate antibiotic.
  2. Grow overnight at 37°C.


Adapted from [http://openwetware.org/wiki/Transforming_chemically_competent_cells_%28Inoue%29 OpenWetWare protocol].

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