Preparing chemically competent cells

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==Using==
==Using==
#Thaw TSS cells on ice.
#Thaw TSS cells on ice.
-
#Add DNA, pipette gently to mix.
+
#Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
-
#Let sit 30 minutes on ice.
+
#Let sit for 30 minutes on ice.
#Incubate cells for 30 seconds at <math>42^o</math>C.
#Incubate cells for 30 seconds at <math>42^o</math>C.
-
#Add 1 mL [[SOC]] (Room Temp).
+
#Add 1 mL [[SOC]] ([[2XYT]] is also suitable) at room temp.
#Incubate for 1 hour at <math>37^o</math>C.
#Incubate for 1 hour at <math>37^o</math>C.
-
#Plate 200 µL onto appropriate resistance plate.
+
#Plate 200 µL onto plate with appropriate antibiotic.

Revision as of 11:31, 8 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 (1μl of prepped plasmid is more than enough).
  3. Let sit for 30 minutes on ice.
  4. Incubate cells for 30 seconds at 42oC.
  5. Add 1 mL SOC (2XYT is also suitable) at room temp.
  6. Incubate for 1 hour at 37oC.
  7. Plate 200 µL onto plate with appropriate antibiotic.


TSS (50 mL)

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