Transforming chemically competent cells (Inoue)

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(Method)
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#Incubate on ice for 30 minutes
#Incubate on ice for 30 minutes
#*Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
#*Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
-
#Incubate cells for 30 seconds at <math>42^o</math>C.
+
#Incubate cells for 30 seconds at 42&deg;C.
#Incubate cells on ice for 2 min.
#Incubate cells on ice for 2 min.
#Add 4 volumes of room temperature [[SOC]] (not critical)
#Add 4 volumes of room temperature [[SOC]] (not critical)
-
#Incubate for 1 hour at <math>37^o</math>C on shaker.
+
#Incubate for 1 hour at 37&deg;C on shaker.
#*Note: Can also save some time here by reducing incubation to ~45 min.
#*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
#*Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics
#Spread 100-300 &mu;l onto a plate made with appropriate antibiotic.
#Spread 100-300 &mu;l onto a plate made with appropriate antibiotic.
-
#Grow overnight at 37 &deg;C.
+
#Grow overnight at 37&deg;C.
[[Category:E.Coli Protocol]]
[[Category:E.Coli Protocol]]

Revision as of 07:28, 12 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.
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