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

Revision as of 11:24, 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 Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle 4^o} C.
  3. Remove supernatant and replace with 10% original volume TSS.
  4. Make 100 uL aliquots and store at Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle -80^o} C.

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 Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle 42^o} C.
  5. Add 1 mL SOC (Room Temp).
  6. Incubate for 1 hour at Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle 37^o} C.
  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