Template:SBB-Protocols Micro1

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Current revision (14:14, 7 June 2010) (view source)
 
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'''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.'''
   1. Thaw a 200 uL aliquot of cells on ice
   1. Thaw a 200 uL aliquot of cells on ice
-
   2. Add 50 uL of water to the cells
+
   2. Add 50 uL of water to the cells (if greater volume is desired)
   3. Add 30 uL of KCM to the cells  
   3. Add 30 uL of KCM to the cells  
-
   4. Put your ligation mixture on ice, let cool a minute or two
+
   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
   5. Add 70 uL of the cell cocktail to the ligation, stir to mix
   6. Let sit on ice for 10 min
   6. Let sit on ice for 10 min

Current revision

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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