User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/01/16: Difference between revisions

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==Protocol==
==Protocol==
# Thaw a tube of BL21(DE3) Competent E. coli cells on ice for 10 minutes
# Thaw a tube of BL21(DE3) Competent E. coli cells on ice for 10 minutes
-Because the obtained cells were at -80 degrees Celsius they were left on ice for longer
##Because the obtained cells were at -80 degrees Celsius they were left on ice for longer
#A stock solution containing the plasmid DNA was made
#A stock solution containing the plasmid DNA was made
##9.6ug of DNA were diluted into 960uL of sterilized water
##9.6ug of DNA were diluted into 960uL of sterilized water
##This solution along with the tube in which the mixture will occur were also placed on ice pending the thawing of the cells.
#Add 1-5uL containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
#Add 1-5uL containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
##1uL of the DNA solution were added to 30uL of cells in the provided tube.
##The mixture was flicked and placed on ice for 30 minutes.
#Place on ice for 30 minutes. Do not mix
#Place on ice for 30 minutes. Do not mix
#Heat shock at exactly 42 degrees Celsius for exactly 10 seconds. Do not mix.
#Heat shock at exactly 42 degrees Celsius for exactly 10 seconds. Do not mix.
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#Spread 50-100uL of each dilution onto a selection plate and incubate overnight at 37degrees Celsius. Alternatively, incubate at 30 degrees celsius for 24-36 hours or at 25 degrees Celsius for 48 hours.
#Spread 50-100uL of each dilution onto a selection plate and incubate overnight at 37degrees Celsius. Alternatively, incubate at 30 degrees celsius for 24-36 hours or at 25 degrees Celsius for 48 hours.


==Notes==
Thawing: Cells are best thawed on ice and DNA added as soon as the last bit of ice in the tube disappears. Cells can also be thawed by hand, but warming above 0 degrees Celsius will decrease the transformation efficiency.


Incubation of DNA with Cells on ice: For maximum transformation efficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes you shorten this step.
Heat Shock: Both the temperature and the timing go the heat shock step are important and specific to the transformation volume and vessel. Using the transformation tube provided, 10 seconds at 42 degrees Celsius in optimal.
Outgrowth: Outgrowth at 37 degrees Celsius for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in transformation efficiency for every 15 minutes you shorten this step. SOC gives 2-fold higher transformation efficiency than LB medium; and incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than incubation without shaking.
Plating: Selection plates can be used warm or cold, wet or dry without significantly affecting the transformation efficiency. However, warm, dry plates are easier to spread and allow for the most rapid colony formation.
==Results==
After 24 hours of incubating, no cells grew on the plate. This is possibly caused by unsuccessful transformation of cells.
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Revision as of 08:01, 23 January 2014

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Cell Transformation

The objective of this session was to insert plasmid DNA into cells in order to transform the cells. The protocol from New England Biolabs for transformation (C2527) was followed.

Protocol

  1. Thaw a tube of BL21(DE3) Competent E. coli cells on ice for 10 minutes
    1. Because the obtained cells were at -80 degrees Celsius they were left on ice for longer
  2. A stock solution containing the plasmid DNA was made
    1. 9.6ug of DNA were diluted into 960uL of sterilized water
    2. This solution along with the tube in which the mixture will occur were also placed on ice pending the thawing of the cells.
  3. Add 1-5uL containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
    1. 1uL of the DNA solution were added to 30uL of cells in the provided tube.
    2. The mixture was flicked and placed on ice for 30 minutes.
  4. Place on ice for 30 minutes. Do not mix
  5. Heat shock at exactly 42 degrees Celsius for exactly 10 seconds. Do not mix.
  6. Place on ice for 5 minutes. Do not mix.
  7. Pipette 950uL of room temperature SOC into the mixture.
  8. Place at 37 degrees Celsius for 60 minutes. Shake vigorously (250 rpm) or rotate.
  9. Warm selection plates to 37 degrees Celsius.
  10. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
  11. Spread 50-100uL of each dilution onto a selection plate and incubate overnight at 37degrees Celsius. Alternatively, incubate at 30 degrees celsius for 24-36 hours or at 25 degrees Celsius for 48 hours.

Notes

Thawing: Cells are best thawed on ice and DNA added as soon as the last bit of ice in the tube disappears. Cells can also be thawed by hand, but warming above 0 degrees Celsius will decrease the transformation efficiency.

Incubation of DNA with Cells on ice: For maximum transformation efficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes you shorten this step.

Heat Shock: Both the temperature and the timing go the heat shock step are important and specific to the transformation volume and vessel. Using the transformation tube provided, 10 seconds at 42 degrees Celsius in optimal.

Outgrowth: Outgrowth at 37 degrees Celsius for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in transformation efficiency for every 15 minutes you shorten this step. SOC gives 2-fold higher transformation efficiency than LB medium; and incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than incubation without shaking.

Plating: Selection plates can be used warm or cold, wet or dry without significantly affecting the transformation efficiency. However, warm, dry plates are easier to spread and allow for the most rapid colony formation.

Results

After 24 hours of incubating, no cells grew on the plate. This is possibly caused by unsuccessful transformation of cells.


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