Template:SBB-Protocols Micro1: Difference between revisions
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#Thaw a 200 uL aliquot of cells on ice | #Thaw a 200 uL aliquot of cells on ice | ||
#Put your ligation mixture on ice | #Put your ligation mixture on ice | ||
#Add | #Add 70 uL of water to the mixture | ||
#Add 20 uL of KCM salts to the mixture | #Add 20 uL of KCM salts to the mixture | ||
#Add | #Add 90-100 uL of the cell cocktail to the ligation, pipette up and down gently to mix | ||
#Let sit on ice for 5 min | #Let sit on ice for 5 min | ||
#Heat shock for 2 min at 42 | #Heat shock for 2 min at 42 | ||
Line 11: | Line 11: | ||
#For ampicillin selection, you can plate immediately, otherwise: | #For ampicillin selection, you can plate immediately, otherwise: | ||
#Add 100uL of LB, let shake in the 37 degree incubator for 40 min | #Add 100uL of LB, let shake in the 37 degree incubator for 40 min | ||
#Plate on selective antibiotics, let incubate overnight | #Plate 70+ uL on selective antibiotics, let incubate overnight |
Revision as of 12:41, 23 June 2009
Transformation by heat-shock
Competent cells are stored as 200uL aliquots in the -80 freezer as a communal stock.
- Thaw a 200 uL aliquot of cells on ice
- Put your ligation mixture on ice
- Add 70 uL of water to the mixture
- Add 20 uL of KCM salts to the mixture
- Add 90-100 uL of the cell cocktail to the ligation, pipette up and down gently to mix
- Let sit on ice for 5 min
- Heat shock for 2 min at 42
- Put back on ice for 1 min
- For ampicillin selection, you can plate immediately, otherwise:
- Add 100uL of LB, let shake in the 37 degree incubator for 40 min
- Plate 70+ uL on selective antibiotics, let incubate overnight