Difference: ProtocolsTestingTransformEff (1 vs. 3)

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META TOPICPARENT name="ProtocolList"

Checking Transformation Efficiency of Chemically Competent Cells

Adapted from Molecular Cloning: A Laboratory Manual 3rd Ed., Sambrook and Russell (2001)
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SUPPLIES:

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SUPPLIES

 
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Equipment:

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Equipment

 
  • Shaking Incubator or Shaking Platform and 1L flask clamps
  • 42C Heating Bath or Block
  • 37C Incubator
  • Colony Counter
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Consumables:

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Consumables

 
  • Agar plates with appropriate antibiotic
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Buffers and Solutions:

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Buffers and Solutions

 
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  • Competent cells prepared using the Inoue method or another method
 
  • 10pg / μL solution of a standard plasmid (e.g. pUC19)
  • SOC Medium
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PROTOCOL:

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PROTOCOL

 
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  1. ) Transform 10pg of a standard plasmid (e.g. pUC19) in 50μL of cells using the standard transformation protocol.
  2. ) Plate 50uL of transformed cells in triplicate on appropriate antibiotic
  3. ) Grow plates overnight, and count colonies the next morning
  4. ) Average colony counts for the three plates; efficiency = (# colonies) x 106 cfu / μg
  5. ) Expected efficiency for the Inoue protocol is 1-3 x 108 colonies / μg of plasmid
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  1. Transform 10 pg of a standard plasmid (e.g. pUC19) in 50 L of cells using the standard transformation protocol.
  2. Plate 50 L of transformed cells in triplicate on appropriate antibiotic
  3. Grow plates overnight, and count colonies the next morning
  4. Average colony counts for the three plates; efficiency = (average # colonies) x 106 cfu / μg
  5. Expected efficiency for chemically competent cells prepared by the Inoue protocol is 1-3 x 108 colonies / μg of plasmid
 
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OTHER RESOURCES

 
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-- Main.KateElston - 25 Oct 2018

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Revision 22018-10-26 - KateElston

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META TOPICPARENT name="ProtocolList"

Checking Transformation Efficiency of Chemically Competent Cells

Adapted from Molecular Cloning: A Laboratory Manual 3rd Ed., Sambrook and Russell (2001)
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PROTOCOL:

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  1. ) Transform 10pg of a standard plasmid (e.g. pUC19) in 50μL of cells using the standard transformation protocol
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  1. ) Transform 10pg of a standard plasmid (e.g. pUC19) in 50μL of cells using the standard transformation protocol.
 
  1. ) Plate 50uL of transformed cells in triplicate on appropriate antibiotic
  2. ) Grow plates overnight, and count colonies the next morning
  3. ) Average colony counts for the three plates; efficiency = (# colonies) x 106 cfu / μg

Revision 12018-10-25 - KateElston

Line: 1 to 1
Added:
>
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META TOPICPARENT name="ProtocolList"

Checking Transformation Efficiency of Chemically Competent Cells

Adapted from Molecular Cloning: A Laboratory Manual 3rd Ed., Sambrook and Russell (2001)

SUPPLIES:

Equipment:

  • Shaking Incubator or Shaking Platform and 1L flask clamps
  • 42C Heating Bath or Block
  • 37C Incubator
  • Colony Counter

Consumables:

  • Agar plates with appropriate antibiotic

Buffers and Solutions:

  • Competent cells prepared using the Inoue method
  • 10pg / μL solution of a standard plasmid (e.g. pUC19)
  • SOC Medium

PROTOCOL:

  1. ) Transform 10pg of a standard plasmid (e.g. pUC19) in 50μL of cells using the standard transformation protocol
  2. ) Plate 50uL of transformed cells in triplicate on appropriate antibiotic
  3. ) Grow plates overnight, and count colonies the next morning
  4. ) Average colony counts for the three plates; efficiency = (# colonies) x 106 cfu / μg
  5. ) Expected efficiency for the Inoue protocol is 1-3 x 108 colonies / μg of plasmid

-- Main.KateElston - 25 Oct 2018

 
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