Team:Northwestern/Notebook/Protocols/Transformation

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#Incubate cells for 40 seconds at 42C (use a stopwatch for exact timing).
#Incubate cells for 40 seconds at 42C (use a stopwatch for exact timing).
#Incubate cells on ice for 2 min.
#Incubate cells on ice for 2 min.
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#Add 200 uL LB ([[2XYT]] and SOC are also suitable for greater efficiency)
+
#Add 200 uL LB.
#Incubate for 1 hour at 37C 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)
#Incubate for 1 hour at 37C 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)
#Spread 200uL onto a plate made with appropriate antibiotic.
#Spread 200uL onto a plate made with appropriate antibiotic.

Revision as of 20:37, 11 July 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).
    • Add 10uL of diH2O. 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 42C (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 37C 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 37C.
  9. Save the rest of the transformants in liquid culture at 4 °C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.


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