Chemically Competent Cells

Preparation of Chemically Competent Cells

There are a few variations on the protocol for preparation of chemically competent cells. Choice of protocol may depend on amount of time involved in the prep and the transformation efficiency of the prepared cells.

• CaCl₂/Glycerol method - "quick and efficient" method
• No-Kit Inoue Chemically Competent Cells

Transforming Chemically Competent Cells

SUPPLIES:

Equipment:

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

Consumables:

• Agar plates with appropriate antibiotic

Buffers and Solutions:

• Competent Cells prepared using one of the methods listed above
• SOC Medium

PROTOCOL:

1. ) Remove one tube of cells from the freezer for each transformation reaction, plus appropriate controls
2. ) Thaw tubes on ice
3. ) Add transforming DNA (up to 25ng, or 0.5-2μL of a miniprepped plasmid) and incubate on ice 30 min.
   • For transforming cloning reactions (e.g. from Gibson assembly or MegaWhop), it is possible to add up to 5μL of reaction directly to the cells without any purification steps
4. ) Transfer tubes to a 42°C heat block or water bath for EXACTLY 90 seconds
   • If using a heat block, make sure to fill the wells with diH₂O to ensure efficient heat transfer
5. ) Immediately transfer tubes to ice, cool 1-2 minutes
6. ) Add 450μL SOC medium to each tube, and shake @ 200-250RPM for 1 hour
   • For tranformations in eppendorf tubes, can just put the tubes inside an empty 250mL flask and incubate in the large shaking incubators
7. ) Plate at least two different volumes (10μL and 50μL usually work well) of each reaction on appropriate antibiotic plates
   • If your efficiencies tend to be on the low side, pellet your cells by centrifuging for 5 min at 3000rpm then resuspend in 100μL and plate the entire mixture

• Note that this protocol can also be done in PCR plates for large numbers of transformations. Aliquot 10μL of cells in each well (this can be done when cells are prepared or with freshly-thawed cells) and add 0.5-1μL of DNA. Incubation steps can be done on the block of a pre-heated or -cooled thermocycler (timing is more exact if you heat the block first and transfer the plate by hand rather than changing temp of the block).

Protocol Contributors:

• Colin Brown
• Matt McGuffie
• Kate Elston

Chemically Competent Cells

Preparation of Chemically Competent Cells

There are a few variations on the protocol for preparation of chemically competent cells. Choice of protocol may depend on amount of time involved in the prep and the transformation efficiency of the prepared cells.

• CaCl₂/Glycerol method - "quick and efficient" method
• No-Kit Inoue Chemically Competent Cells

Transforming Chemically Competent Cells

SUPPLIES:

Equipment:

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

Consumables:

• Agar plates with appropriate antibiotic

Buffers and Solutions:

• Competent Cells prepared using one of the methods listed above
• SOC Medium

PROTOCOL:

1. ) Remove one tube of cells from the freezer for each transformation reaction, plus appropriate controls
2. ) Thaw tubes on ice
3. ) Add transforming DNA (up to 25ng, or 0.5-2μL of a miniprepped plasmid) and incubate on ice 30 min.
   • For transforming cloning reactions (e.g. from Gibson assembly or MegaWhop), it is possible to add up to 5μL of reaction directly to the cells without any purification steps
4. ) Transfer tubes to a 42°C heat block or water bath for EXACTLY 90 seconds
   • If using a heat block, make sure to fill the wells with diH₂O to ensure efficient heat transfer
5. ) Immediately transfer tubes to ice, cool 1-2 minutes

• • For tranformations in eppendorf tubes, can just put the tubes inside an empty 250mL flask and incubate in the large shaking incubators

1. ) Plate at least two different volumes (10μL and 50μL usually work well) of each reaction on appropriate antibiotic plates
   • If your efficiencies tend to be on the low side, pellet your cells by centrifuging for 5 min at 3000rpm then resuspend in 100μL and plate the entire mixture

• Note that this protocol can also be done in PCR plates for large numbers of transformations. Aliquot 10μL of cells in each well (this can be done when cells are prepared or with freshly-thawed cells) and add 0.5-1μL of DNA. Incubation steps can be done on the block of a pre-heated or -cooled thermocycler (timing is more exact if you heat the block first and transfer the plate by hand rather than changing temp of the block).

Protocol Contributors:

• Colin Brown
• Matt McGuffie
• Kate Elston

Chemically Competent Cells

Preparation of Chemically Competent Cells

There are a few variations on the protocol for preparation of chemically competent cells. Choice of protocol may depend on amount of time involved in the prep and the transformation efficiency of the prepared cells.

• No-Kit Inoue Chemically Competent Cells

Transforming Chemically Competent Cells

SUPPLIES:

Equipment:

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

Consumables:

• Agar plates with appropriate antibiotic

Buffers and Solutions:

• SOC Medium

PROTOCOL:

1. ) Remove one tube of cells from the freezer for each transformation reaction, plus appropriate controls

• • For transforming cloning reactions (e.g. from Gibson assembly or MegaWhop), it is possible to add up to 5μL of reaction directly to the cells without any purification steps

1. ) Transfer tubes to a 42°C heat block or water bath for EXACTLY 90 seconds
   • If using a heat block, make sure to fill the wells with diH₂O to ensure efficient heat transfer
2. ) Immediately transfer tubes to ice, cool 1-2 minutes

• • For tranformations in eppendorf tubes, can just put the tubes inside an empty 250mL flask and incubate in the large shaking incubators

1. ) Plate at least two different volumes (10μL and 50μL usually work well) of each reaction on appropriate antibiotic plates

• Note that this protocol can also be done in PCR plates for large numbers of transformations. Aliquot 10μL of cells in each well (this can be done when cells are prepared or with freshly-thawed cells) and add 0.5-1μL of DNA. Incubation steps can be done on the block of a pre-heated or -cooled thermocycler (timing is more exact if you heat the block first and transfer the plate by hand rather than changing temp of the block).

Protocol Contributors:

• Colin Brown
• Matt McGuffie
• Kate Elston

Chemically Competent Cells

Preparation of Chemically Competent Cells

There are a few variations on the protocol for preparation of chemically competent cells. Choice of protocol may depend on amount of time involved in the prep and the transformation efficiency of the prepared cells.

• No-Kit Inoue Chemically Competent Cells
• CaCl₂/Glycerol method - "quick and efficient" method

Transforming Chemically Competent Cells

SUPPLIES:

Equipment:

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

Consumables:

• Agar plates with appropriate antibiotic

Buffers and Solutions:

• Competent Cells prepared using the Inoue method

PROTOCOL:

1. ) Remove one tube of cells from the freezer for each transformation reaction, plus appropriate controls
2. ) Quickly thaw tubes by holding in your hand for a few seconds
3. ) Add transforming DNA (up to 25ng, or 0.5-1uL of a miniprepped plasmid) and incubate on ice 30 min.
   • For transforming cloning reactions (e.g. from Gibson assembly or MegaWhop), it is possible to add up to 5μL of reaction directly to the cells without any purification steps
4. ) Transfer tubes to a 42°C heat block or water bath for EXACTLY 90 seconds
   • If using a heat block, make sure to fill the wells with diH₂O to ensure efficient heat transfer
5. ) Immediately transfer tubes to ice, cool 1-2 minutes
6. ) Add 450μL SOC medium to each tube, and shake @200-250RPM for 45min
   • For tranformations in eppendorf tubes, can just put the tubes inside an empty 250mL flask and incubate in the large shaking incubators
7. ) Plate at least two different volumes (10μL and 50μL usually work well) of each reaction on appropriate antibiotic plates

• Note that this protocol can also be done in PCR plates for large numbers of transformations. Aliquot 10μL of cells in each well (this can be done when cells are prepared or with freshly-thawed cells) and add 0.5-1μL of DNA. Incubation steps can be done on the block of a pre-heated or -cooled thermocycler (timing is more exact if you heat the block first and transfer the plate by hand rather than changing temp of the block).

Protocol Contributors:

• Colin Brown
• Matt McGuffie
• Kate Elston

Chemically Competent Cells

Preparation of Chemically Competent Cells

There are a few variations on the protocol for preparation of chemically competent cells. Choice of protocol may depend on amount of time involved in the prep and the transformation efficiency of the prepared cells.

• No-Kit Inoue Chemically Competent Cells
• CaCl₂/Glycerol method - "quick and efficient" method

Transforming Chemically Competent Cells

SUPPLIES:

Equipment:

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

Consumables:

• Agar plates with appropriate antibiotic

Buffers and Solutions:

• Competent Cells prepared using the Inoue method
• SOC Medium

PROTOCOL:

1. ) Remove one tube of cells from the freezer for each transformation reaction, plus appropriate controls
2. ) Quickly thaw tubes by holding in your hand for a few seconds
3. ) Add transforming DNA (up to 25ng, or 0.5-1uL of a miniprepped plasmid) and incubate on ice 30 min.
   • For transforming cloning reactions (e.g. from Gibson assembly or MegaWhop), it is possible to add up to 5μL of reaction directly to the cells without any purification steps
4. ) Transfer tubes to a 42°C heat block or water bath for EXACTLY 90 seconds
   • If using a heat block, make sure to fill the wells with diH₂O to ensure efficient heat transfer
5. ) Immediately transfer tubes to ice, cool 1-2 minutes
6. ) Add 450μL SOC medium to each tube, and shake @200-250RPM for 45min
   • For tranformations in eppendorf tubes, can just put the tubes inside an empty 250mL flask and incubate in the large shaking incubators
7. ) Plate at least two different volumes (10μL and 50μL usually work well) of each reaction on appropriate antibiotic plates

• Note that this protocol can also be done in PCR plates for large numbers of transformations. Aliquot 10μL of cells in each well (this can be done when cells are prepared or with freshly-thawed cells) and add 0.5-1μL of DNA. Incubation steps can be done on the block of a pre-heated or -cooled thermocycler (timing is more exact if you heat the block first and transfer the plate by hand rather than changing temp of the block).

Protocol Contributors:

• Colin Brown
• Matt McGuffie
• Kate Elston