Difference between revisions of "Help:Protocols/Transformation"

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=Transforming Competent Cells=
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'''Estimated time: 3 hours''' (plus 12-14 hour incubation)<br>
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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.
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<td><div id="splash-title">Transformation</div></td>
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Transformations are essential to using the distribution kits sent out by the Registry. They can also be one of the more fickle laboratory techniques. We recommend the following protocol as it is the same one used at iGEM HQ.
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=Transformation Protocol=
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'''Estimated time: 3 hours''' (plus 14-18 hour incubation)<br>
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We have tested transformations of the distribution kit with this protocol and have found that it is the best protocol to use with Registry 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.
  
  
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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'')
 
*[[Help:Spring_2010_DNA_distribution | Resuspended DNA]] (''Resuspend well in 10ul dH20, pipette up and down several times, let sit for a few minutes'')
 
*[[Help:Competent_Cells_Protocol | Competent cells]] (''50ul per transformation'')
 
*[[Help:Competent_Cells_Protocol | Competent cells]] (''50ul per transformation'')
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*Ice
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*Ice (in ice bucket/container)
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*42º water bath  
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*2ml tube (1 per a transformation, chilled in ice)
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*37º incubator
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*42ºC water bath  
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*SOC (''check for contamination!'')
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*37ºC incubator
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*SOC media (''check for contamination!'')
 
*Petri dishes with LB agar and appropriate antibiotic (''two per transformation'')
 
*Petri dishes with LB agar and appropriate antibiotic (''two per transformation'')
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*glass beads or spreader
  
 
==Protocol==
 
==Protocol==
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# Start thawing the competent cells on crushed ice.  
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# Start thawing the competent cells on ice.  
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# 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.
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# Add 50 µL of thawed competent cells into pre-chilled 2ml tube.
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# Incubate the cells on ice for 30 minutes.
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# Add 1 - 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice.
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# Close tube and incubate the cells on ice for 30 minutes.
 
# 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 and is not contaminated)
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# Add 200 μl of SOC media (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.  '''Important: 2 hour recovery time 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 plasmid backbones with antibiotic resistance other than ampicillin.'''
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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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# Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, 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.
 
# 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.
 
# 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.
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# You can pick a single colony, grow up a cell culture and make a glycerol stock.

Revision as of 17:53, 17 May 2012

Transformation Protocol

Estimated time: 3 hours (plus 14-18 hour incubation)
We have tested transformations of the distribution kit with this protocol and have found that it is the best protocol to use with Registry 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 transformation)
  • Ice (in ice bucket/container)
  • 2ml tube (1 per a transformation, chilled in ice)
  • 42ºC water bath
  • 37ºC incubator
  • SOC media (check for contamination!)
  • Petri dishes with LB agar and appropriate antibiotic (two per transformation)
  • glass beads or spreader

Protocol

  1. Start thawing the competent cells on ice.
  2. Add 50 µL of thawed competent cells into pre-chilled 2ml tube.
  3. Add 1 - 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice.
  4. Close tube and incubate the cells on ice for 30 minutes.
  5. 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.
  6. Incubate the cells on ice for 5 minutes.
  7. Add 200 μl of SOC media (make sure that the broth does not contain antibiotics and is not contaminated)
  8. 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 plasmid backbones with antibiotic resistance other than ampicillin.
  9. Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, 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.
  10. 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.
  11. You can pick a single colony, grow up a cell culture and make a glycerol stock.