Team:Northwestern/Notebook/Protocols/Transformation

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#Thaw cells on ice. While thawing, prepare DNA from well plates:
#Thaw 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 Biobrick™-standard 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 Biobrick™-standard 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).
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#*Add 10uL of diH2O (deionized water)
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#*Add 10uL of diH2O
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#Pipette 25uL aliquots into tubes on ice. Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
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#Pipette 25uL aliquots into tubes on ice. Add 1 uL DNA, pipette gently to mix
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#*Note: If you are adding small volumes (~1μl), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.
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#Let sit for 5 minutes on ice.
#Let sit for 5 minutes on ice.
#*Note: For better efficiency do at least 30 minutes
#*Note: For better efficiency do at least 30 minutes

Revision as of 20:29, 11 July 2011

RETURN TO IGEM 2010

Transformation



  1. Thaw 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 Biobrick™-standard 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
  2. Pipette 25uL aliquots into tubes on ice. Add 1 uL DNA, pipette gently to mix
  3. Let sit for 5 minutes on ice.
    • Note: For better efficiency do at least 30 minutes
  4. Incubate cells for 45 seconds at 42C.
  5. Incubate cells on ice for 2 min.
  6. Add 200 uL LB (2XYT and SOC are also suitable for greater efficiency)
  7. Incubate for 1 hour at 37C (for amp you can incubate for 5-10 minutes if you are in a hurry, for other ABs, can probably get away with ~40 minutes)
  8. Spread 100-300 μl onto a plate made with appropriate antibiotic.
  9. Grow overnight at 37C.
  10. 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]