Team:Northwestern/Notebook/Protocols/Transformation

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#Thaw chemically competent cells on ice. While thawing, prepare DNA from well plates:
#Thaw chemically competent cells on ice. While thawing, prepare DNA from well plates:
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#*With a pipette tip, punch a hole through the foil cover into the corresponding well to the part that you want. Make sure you have properly oriented the plate (label rows/columns with a sharpie if it's the first time using the plate).
+
#*With a pipette tip, punch a hole through the foil cover into the corresponding well to the part that you want. Make sure you have properly oriented the plate (label rows/columns with a sharpie if it's the first time using the plate). Transfer excess DNA from well to a microcentrifuge tube and store in -20C.
#*Add 10uL of nuclease free H2O. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended.  
#*Add 10uL of nuclease free H2O. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended.  
#Pipette 25uL aliquots of cells into tubes on ice. Add 1 uL DNA, then pipette gently to mix. Let sit for at least 30 minutes on ice.  
#Pipette 25uL aliquots of cells into tubes on ice. Add 1 uL DNA, then pipette gently to mix. Let sit for at least 30 minutes on ice.  

Revision as of 18:31, 12 August 2011

RETURN TO IGEM 2010


Transformation



  1. Thaw chemically competent cells on ice. While thawing, prepare DNA from well plates:
    • With a pipette tip, punch a hole through the foil cover into the corresponding well to the part that you want. Make sure you have properly oriented the plate (label rows/columns with a sharpie if it's the first time using the plate). Transfer excess DNA from well to a microcentrifuge tube and store in -20C.
    • Add 10uL of nuclease free H2O. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended.
  2. Pipette 25uL aliquots of cells into tubes on ice. Add 1 uL DNA, then pipette gently to mix. Let sit for at least 30 minutes on ice.
  3. Incubate cells for 40 seconds at 42 °C (use a stopwatch for exact timing).
  4. Incubate cells on ice for 2 min.
  5. Add 200 uL LB.
  6. Incubate for 1 hour at 37 °C in shaker. If you need to cut corners, for Amp you can incubate for 5-10 minutes if you are in a hurry. For other ABs, can probably get away with about 40 minutes).
  7. Spread 200uL onto a plate made with appropriate antibiotic.
  8. Grow overnight at 37 °C.


Adapted from [http://openwetware.org/wiki/Transforming_chemically_competent_cells OpenWetWare] and [http://partsregistry.org/Help:Spring_2011_DNA_distribution The Parts Registry]