Difference: ProtocolsTestingTransformEff (1 vs. 3)

Revision 32021-07-05 - JeffreyBarrick

  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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+ name="ProtocolList"
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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
  42°C Heating Bath or Block
  37°C 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
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+ Competent cells prepared using the Inoue method or another method
 pg / 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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+ ) Transform 10pg of a standard plasmid (e.g. pUC19) in 50μL of cells using the standard transformation protocol.
  ) Plate 50uL of transformed cells in triplicate on appropriate antibiotic
  ) Grow plates overnight, and count colonies the next morning
  ) Average colony counts for the three plates; efficiency = (# colonies) x 10⁶ cfu / μg
  ) Expected efficiency for the Inoue protocol is 1-3 x 10⁸ colonies / μg of plasmid
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+ Transform 10 pg of a standard plasmid (e.g. pUC19) in 50 µL of cells using the standard transformation protocol.
  Plate 50 µL of transformed cells in triplicate on appropriate antibiotic
  Grow plates overnight, and count colonies the next morning
  Average colony counts for the three plates; efficiency = (average # colonies) x 10⁶ cfu / μg
  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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+ iGEM protocol for testing chemically competent cells – describes a similar procedure in more depth.
  NEBalpha competent cells – has some tables showing the effects of changing the amount of plasmid transformed and various other parameters.
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+-- Main.KateElston - 25 Oct 2018

Revision 22018-10-26 - KateElston

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
42°C Heating Bath or Block
37°C 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:

Changed:

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<

) Transform 10pg of a standard plasmid (e.g. pUC19) in 50μL of cells using the standard transformation protocol

>
>

) Transform 10pg of a standard plasmid (e.g. pUC19) in 50μL of cells using the standard transformation protocol.

) Plate 50uL of transformed cells in triplicate on appropriate antibiotic
) Grow plates overnight, and count colonies the next morning
) Average colony counts for the three plates; efficiency = (# colonies) x 10⁶ cfu / μg
) Expected efficiency for the Inoue protocol is 1-3 x 10⁸ colonies / μg of plasmid

-- Main.KateElston - 25 Oct 2018

Revision 12018-10-25 - KateElston

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
42°C Heating Bath or Block
37°C 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:

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

-- Main.KateElston - 25 Oct 2018

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