Template:SBB-Protocols Micro1: Difference between revisions

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===Transformation by heat-shock===
===Transformation by heat-shock===
'''Competent cells are stored as 200uL aliquots in the -80 freezer as a communal stock.'''
'''Competent cells are stored as 200uL aliquots in the -80 freezer as a communal stock.'''
#Thaw a 200 uL aliquot of cells on ice
  1. Thaw a 200 uL aliquot of cells on ice
#Put your ligation mixture on ice
  2. Add 50 uL of water to the cells (if greater volume is desired)
#Add 50 uL of water to the mixture
  3. Add 30 uL of KCM to the cells
#Add 20 uL of KCM salts to the mixture
  4. Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
#Add 75-100 uL of the cell cocktail to the ligation, pipette up and down gently to mix
  5. Add 70 uL of the cell cocktail to the ligation, stir to mix
#Let sit on ice for 5 min
  6. Let sit on ice for 10 min
#Heat shock for 2 min at 42
  7. Heat shock for 90 seconds at 42 (longer incubation may work better)
#Put back on ice for 1 min
  8. Put back on ice for 1 min
#For ampicillin selection, you can plate immediately, otherwise:
  9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
#Add 100uL of LB, let shake in the 37 degree incubator for 40 min
  10. Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight
#Plate on selective antibiotics, let incubate overnight

Latest revision as of 11:14, 7 June 2010

Transformation by heat-shock

Competent cells are stored as 200uL aliquots in the -80 freezer as a communal stock. 

  1. Thaw a 200 uL aliquot of cells on ice
  2. Add 50 uL of water to the cells (if greater volume is desired)
  3. Add 30 uL of KCM to the cells 
  4. Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
  5. Add 70 uL of the cell cocktail to the ligation, stir to mix
  6. Let sit on ice for 10 min
  7. Heat shock for 90 seconds at 42 (longer incubation may work better)
  8. Put back on ice for 1 min
  9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
 10. Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight
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