Difference between revisions of "Preparing chemically competent cells"

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m (Use)
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#Plate 200 µL onto plate with appropriate antibiotic.
 
#Plate 200 µL onto plate with appropriate antibiotic.
  
 
+
==Buffers==
 
[[TSS]] (50 mL)
 
[[TSS]] (50 mL)
 
*5g PEG 8000
 
*5g PEG 8000

Revision as of 13:28, 5 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 [math]4^o[/math]C.
  3. Remove supernatant and replace with 10% original volume TSS.
  4. Make 100 uL aliquots and store at [math]-80^o[/math]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 [math]42^o[/math]C.
  5. Add 1 mL SOC (2XYT is also suitable) at room temp.
  6. Incubate for 1 hour at [math]37^o[/math]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