Difference between revisions of "Preparing chemically competent cells"

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#Let sit for 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]] ([[2XYT]] is also suitable) at room temp.
+
##Note: According to the original TSS paper and qualitative experience, this step is completely optional and may actually reduce transformation efficiency.
 +
#Add 1 mL [[SOC]] ([[2XYT]] and LB are 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 plate with appropriate antibiotic.
 
#Plate 200 µL onto plate with appropriate antibiotic.

Revision as of 11:28, 9 July 2005

Preparation

  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.

Use

  1. Thaw TSS cells on ice.
  2. Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
    1. Note: If you are adding small volumes (~1μl), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.
  3. Let sit for 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.
    1. Note: According to the original TSS paper and qualitative experience, this step is completely optional and may actually reduce transformation efficiency.
  5. Add 1 mL SOC (2XYT and LB are also suitable) at 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 plate with appropriate antibiotic.

Buffers

TSS (50 mL)

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