Difference between revisions of "Help:Transformation Protocol"

(New page: We have found that the following protocol ensures a high efficiency for transforming competent cells. This may be particularly useful if you are finding that your transformations are not w...)
 
 
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We have found that the following protocol ensures a high efficiency for transforming competent cells. This may be particularly useful if you are finding that your transformations are not working, or yielding few colonies.
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#REDIRECT [[Help:Protocols/Transformation]]
  
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*Allow the cells to thaw
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=Transforming Competent Cells=
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*Transfer 50ul into a 1.5 or 2ml centrifuge tube. Transform the 50ul of cells with 1ul of DNA  
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'''Estimated time: 3 hours''' (plus 12-14 hour incubation)<br>
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*Incubate on ice for 30 minutes
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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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*Heat shock for 1 minute at 42C
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*Hold on ice for 5 minutes
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*Add 200 μl of SOC
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==Materials needed==
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*Incubate at 37 C for 2 hours on rotor
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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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*Plate 20 μl and 200ul of the transformation on the appropriate antibiotic plates, and spread using sterile 3.5 mm glass beads
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*[[Help:Competent_Cells_Protocol | Competent cells]] (''50ul per a transformation'')
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*Ice
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*42º water bath
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*37º incubator
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*SOC (''check for contamination!'')
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*Petri dishes with LB agar and appropriate antibiotic (''two per a transformation'')
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==Protocol==
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# Start thawing the competent cells on crushed 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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# Incubate the cells on ice for 30 minutes.
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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.
 +
# Incubate the cells on ice for 5 minutes.
 +
# 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.  '''Important: 2 hour recovery time helps in transformation efficiency, especially for plasmids 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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# 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

Redirect to:

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.