Transformation Protocol: Difference between revisions

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>YanWu
(Created page with "#Ensure water bath at 42 C, warm SOC media in incubator at 37 C #Add 200 ng DNA into each tube of 100 uL cells (1-5 uL vector) #Incubate on ice for 30 minutes #Heat-shock for ...")
 
>YanWu
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#Ensure water bath at 42 C, warm SOC media in incubator at 37 C
#Ensure water bath at 42 C, warm SOC media in incubator at 37 C
#Add 200 ng DNA into each tube of 100 uL cells (1-5 uL vector)
#Add 200 ng DNA into each tube of 100 uL cells (1-5 uL vector). You want to use up all of your vector so as to get the best coverage.
#Incubate on ice for 30 minutes
#Incubate on ice for 30 minutes
#Heat-shock for 45 seconds @ 42 C
#Heat-shock for 45 seconds @ 42 C

Revision as of 22:55, 6 May 2015

  1. Ensure water bath at 42 C, warm SOC media in incubator at 37 C
  2. Add 200 ng DNA into each tube of 100 uL cells (1-5 uL vector). You want to use up all of your vector so as to get the best coverage.
  3. Incubate on ice for 30 minutes
  4. Heat-shock for 45 seconds @ 42 C
  5. Incubate on ice again for 2 minutes
  6. Rescue cells with 500 uL SOC per tube
  7. Shake at 37 C for 1 hr in incubator
  8. Spread up to 20 uL on at least 2 plates for each library (including control)
  9. Add all tubes into large culture flask (150 mL) grow overnight