Template:SBB-Protocols SmallScaleCompetent

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(New page: #Grow cells in ~4 mL LB until cloudy (OD600=0.5) #Put on ice #Transfer 1mL into an eppendorf tube on ice, let cool #Centrifuge full speed for 30 sec, toss out supernatant #Resuspend in 90u...)
Current revision (14:43, 1 July 2010) (view source)
 
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#Grow cells in ~4 mL LB until cloudy (OD600=0.5)
+
#Grow 30uL of cells in ~4 mL LB until cloudy (OD600=0.5)
#Put on ice
#Put on ice
#Transfer 1mL into an eppendorf tube on ice, let cool
#Transfer 1mL into an eppendorf tube on ice, let cool

Current revision

  1. Grow 30uL of cells in ~4 mL LB until cloudy (OD600=0.5)
  2. Put on ice
  3. Transfer 1mL into an eppendorf tube on ice, let cool
  4. Centrifuge full speed for 30 sec, toss out supernatant
  5. Resuspend in 90uL of TSS solution
  6. Add 10uL KCM
  Step 6.5:  do the negative control before adding the DNA
  1. Add 1uL plasmid DNA
  2. Let sit on ice for 10min, heat shock 90 sec at 42, ice for a minute, rescue 1 hr, then incubate and/or plate


        • Always do negative controls on your competent cell prep!

Sector a region of your plate and spread about 5uL of untransformed competent cells to confirm that they are free of contamination.

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