Difference between revisions of "Help:Transformation Protocol"
 
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#REDIRECT [[Help:Protocols/Transformation]]
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=Transforming Competent Cells=
 
=Transforming Competent Cells=
 
'''Estimated time: 3 hours''' (plus 12-14 hour incubation)<br>
 
'''Estimated time: 3 hours''' (plus 12-14 hour incubation)<br>
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==Materials needed==
 
==Materials needed==
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*Resuspended DNA (''Resuspend well in 15ul TE or dH20, pipette up and down several times, let sit for a few minutes'')
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*[[Help:Spring_2010_DNA_distribution | Resuspended DNA]] (''Resuspend well in 10ul dH20, pipette up and down several times, let sit for a few minutes'')
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*Competent cells (''50ul per a transformation'')
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*[[Help:Competent_Cells_Protocol | Competent cells]] (''50ul per a transformation'')
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*1.5 - 2.0ml eppendorf tube (''one per a transformation'')
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*Ice
 
*Ice
 
*42º water bath  
 
*42º water bath  
 
*37º incubator
 
*37º incubator
 
*SOC (''check for contamination!'')
 
*SOC (''check for contamination!'')
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*Petri dishes with LB agar and appropriate antibiotic (''two per a transformation'')
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*Petri dishes with LB agar and appropriate antibiotic (''two per a transformation'')
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==Protocol==
 
==Protocol==
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# Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.  A water bath improves heat transfer to the cells.
 
# Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.  A water bath improves heat transfer to the cells.
 
# Incubate the cells on ice for 5 minutes.
 
# Incubate the cells on ice for 5 minutes.
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# Add 200 μl of SOC broth (make sure that the broth does not contain antibiotics or is contaminated)
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# Add 200 μl of SOC broth (make sure that the broth does not contain antibiotics and is not contaminated)
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# Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking.  We have found that growth for 2 hours helps in transformation efficiency, especially for plasmids with antibiotic resistance other than ampicillin.
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# Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking.  '''Important: 2 hour recovery time helps in transformation efficiency, especially for plasmids with antibiotic resistance other than ampicillin.'''
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# Label two LB agar plates containing the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance.  Plate 20 µl and 200 µl of the transformation on the plates. This helps ensure that you will be able to pick out a single colony.
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# Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance.  Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.
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# Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up.  If incubated for too long the antibiotics, especially ampicillin, start to break down and un-transformed cells will begin to grow.
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# Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up.  If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivate the antibiotic outside of the bacteria.

Latest revision as of 18:55, 27 May 2010

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Transforming Competent Cells

Estimated time: 3 hours (plus 12-14 hour incubation)
It is important to note that we have tested transformations of the distribution kit with this protocol. We have found that it is the best protocol to use with BioBrick parts and ensures the highest efficiency for the transformation. This protocol may be particularly useful if you are finding that your transformations are not working, or yielding few colonies.


Materials needed

  • Resuspended DNA (Resuspend well in 10ul dH20, pipette up and down several times, let sit for a few minutes)
  • Competent cells (50ul per a transformation)
  • Ice
  • 42º water bath
  • 37º incubator
  • SOC (check for contamination!)
  • Petri dishes with LB agar and appropriate antibiotic (two per a transformation)

Protocol

  1. Start thawing the competent cells on crushed ice.
  2. Add 50 µL of thawed competent cells and then 1 - 2 µL of the resuspended DNA to the labelled tubes. Make sure to keep the competent cells on ice.
  3. Incubate the cells on ice for 30 minutes.
  4. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds. A water bath improves heat transfer to the cells.
  5. Incubate the cells on ice for 5 minutes.
  6. Add 200 μl of SOC broth (make sure that the broth does not contain antibiotics and is not contaminated)
  7. Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmids with antibiotic resistance other than ampicillin.
  8. Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.
  9. Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivate the antibiotic outside of the bacteria.