Difference between revisions of "TSS Competent Cells"

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#Grow cells in ~4 mL LB until cloudy (OD600=0.5)
+
#Grow cells in ~4 mL LB until cloudy (OD<sub>600</sub> = 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
 
#Centrifuge full speed for 30 sec, toss out supernatant
 
#Centrifuge full speed for 30 sec, toss out supernatant
 
#Resuspend in 90uL of [http://openwetware.org/wiki/TSS TSS buffer]
 
#Resuspend in 90uL of [http://openwetware.org/wiki/TSS TSS buffer]
#Add 10uL [http://gcat.davidson.edu/GcatWiki/index.php/KCM_Preparation KCM]
+
#Add 10uL KCM
 
#Spread about 5uL of untransformed competent cells to confirm that they are free of contamination.
 
#Spread about 5uL of untransformed competent cells to confirm that they are free of contamination.
 
#Add 1uL of DNA (use more [5uL] after a ligation)
 
#Add 1uL of DNA (use more [5uL] after a ligation)
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==5X KCM==
 
==5X KCM==
 
#0.5M KCl
 
#0.5M KCl
#0.15M CaCl2
+
#0.15M CaCl<sub>2</sub>
#0.25M MgCl2
+
#0.25M MgCl<sub>2</sub>

Latest revision as of 14:51, 19 October 2011

  1. Grow 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 buffer
  6. Add 10uL KCM
  7. Spread about 5uL of untransformed competent cells to confirm that they are free of contamination.
  8. Add 1uL of DNA (use more [5uL] after a ligation)
  9. Let sit on ice for 10min
  10. Heat shock 90 sec at 42
  11. Rescue for 1 hour in 100 uL of LB if antibiotic is not Amp.
  12. Plate


5X KCM

  1. 0.5M KCl
  2. 0.15M CaCl2
  3. 0.25M MgCl2