Team:Washington/Protocols/Elect.

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# Add 1μL of a Gibson Product into each tube
# Add 1μL of a Gibson Product into each tube
# Using the Electroporator:
# Using the Electroporator:
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## Set the electroporator to 1250 V. and press "time constant".
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#* Set the electroporator to 1250 V. and press "time constant".
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## Obtain a chilled cuvette, and pipet all 40 uL of a sample into the center.
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#* Obtain a chilled cuvette, and pipet all 40 uL of a sample into the center.
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## Place the cuvette into the electroporator and press "?????????".  
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#* Place the cuvette into the electroporator and press "Pluse" twice.  
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## Immediately remove the cuvette and rescue the sample in 300-500 mL of LB.  
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#* Immediately remove the cuvette and rescue the sample in 300-500 mL of LB.  
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## Pipet the sample into a labeled microcentrifuge tube, and record the time constant. (The time constant should have a value '''greater than 2.5)'''.
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#* Pipet the sample into a labeled microcentrifuge tube, and record the time constant. (The time constant should have a value '''greater than 2.5)'''.
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##Repeat this process for the other sample. After the addition of LB, transfer the sample into another labeled microcentrifuge tube.  
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#** Repeat this process for the other sample. After the addition of LB, transfer the sample into another labeled microcentrifuge tube.  
# Incubate both samples for ~1 hour @ 37oC.  
# Incubate both samples for ~1 hour @ 37oC.  
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## After incubation, combine both samples (make sure each sample has a comparable time constant) and follow standard plating procedures.
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#* After incubation, combine both samples (make sure each sample has a comparable time constant) and follow standard plating procedures.

Latest revision as of 18:31, 14 September 2011


Electroporation Protocol

  1. Obtain a competent cell aliquot from the -80oC freezer, keep the tubes on ice.
  2. Add 40μL of ice cold H2O to each aliquot to make a 80 μL solution.
  3. Separate the 80 uL solution into 2 separate tubes to make two 40uL tubes of solution.
  4. Add 1μL of a Gibson Product into each tube
  5. Using the Electroporator:
    • Set the electroporator to 1250 V. and press "time constant".
    • Obtain a chilled cuvette, and pipet all 40 uL of a sample into the center.
    • Place the cuvette into the electroporator and press "Pluse" twice.
    • Immediately remove the cuvette and rescue the sample in 300-500 mL of LB.
    • Pipet the sample into a labeled microcentrifuge tube, and record the time constant. (The time constant should have a value greater than 2.5).
      • Repeat this process for the other sample. After the addition of LB, transfer the sample into another labeled microcentrifuge tube.
  6. Incubate both samples for ~1 hour @ 37oC.
    • After incubation, combine both samples (make sure each sample has a comparable time constant) and follow standard plating procedures.
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