SUPPLIES

Equipment

Consumables

Buffers and Solutions

• Competent cells prepared using the Inoue method or another method

PROTOCOL

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

OTHER RESOURCES

• 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.

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

Checking Transformation Efficiency of Chemically Competent Cells

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:

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