Difference between revisions of "Team:CCA San Diego/protocols"

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Protocols

Here we can talk about protocols in the lab

Transformation with Digest

Materials

DNA miniprrep

LB agar plates, Cat No. Teknova, appropriate antibiotic

DH5a competent cells, Invitrogen, Cat 18265-017

SOC (Recovery Medium), Lucigen, Cat No. F98226

1.5 mL tube

Vortex

Pipet and tips

Ice bucket and ice

Water bath (42°C)

Incubator (37°C) and shaker

Methods

Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells

For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells

Turn on incubator-shaker at 37°C.

Turn on incubator for plates at 37°C.

Set up water bath at 42°C.

Bring to room temperature S.O.C medium.

Bring LB plates supplemented with appropriate antibiotic at room temperature.

Thaw competent cells on ice.

Aliquots competent cells in as many tubes as needed.

Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix

Incubate on ice for 20 minutes

Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.

Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube

Incubate in shaker at 37°C, 225 rpm for 30 min

Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)

Incubate plates at 37°C overnight

Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

Digest Ligations

Materials

T4 DNA Ligation Buffer. Invitrogen, Cat No. 46-0114

T4 DNA Ligase, Thermo Scientific, Cat No. K1231

Reagent grade water, NERL, Cat No. 98555

1.5 mL tube

Vortex

Pipet and tips

Ice bucket and ice

Methods

Set ligation as shown below in 1.5 mL tube.

The tubes were incubated at room temperature for 1 hour.

Turn on incubator-shaker at 37°C.

The tubes were then transferred to ice.

Ligation Condition (with insert)

Bring to room temperature S.O.C medium.

Bring LB plates supplemented with appropriate antibiotic at room temperature.

Thaw competent cells on ice.

Aliquots competent cells in as many tubes as needed.

Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix

Incubate on ice for 20 minutes

Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.

Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube

Incubate in shaker at 37°C, 225 rpm for 30 min

Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)

Incubate plates at 37°C overnight

Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

OD Calibration

Materials

DNA miniprrep

LB agar plates, Cat No. Teknova, appropriate antibiotic

DH5a competent cells, Invitrogen, Cat 18265-017

SOC (Recovery Medium), Lucigen, Cat No. F98226

1.5 mL tube

Vortex

Pipet and tips

Ice bucket and ice

Water bath (42°C)

Incubator (37°C) and shaker

Methods

Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells

For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells

Turn on incubator-shaker at 37°C.

Turn on incubator for plates at 37°C.

Set up water bath at 42°C.

Bring to room temperature S.O.C medium.

Bring LB plates supplemented with appropriate antibiotic at room temperature.

Thaw competent cells on ice.

Aliquots competent cells in as many tubes as needed.

Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix

Incubate on ice for 20 minutes

Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.

Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube

Incubate in shaker at 37°C, 225 rpm for 30 min

Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)

Incubate plates at 37°C overnight

Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

Biotransformation Measurement

Materials

DNA miniprrep

LB agar plates, Cat No. Teknova, appropriate antibiotic

DH5a competent cells, Invitrogen, Cat 18265-017

SOC (Recovery Medium), Lucigen, Cat No. F98226

1.5 mL tube

Vortex

Pipet and tips

Ice bucket and ice

Water bath (42°C)

Incubator (37°C) and shaker

Instrumental Methods

Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells

For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells

Turn on incubator-shaker at 37°C.

Turn on incubator for plates at 37°C.

Set up water bath at 42°C.

Bring to room temperature S.O.C medium.

Bring LB plates supplemented with appropriate antibiotic at room temperature.

Thaw competent cells on ice.

Aliquots competent cells in as many tubes as needed.

Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix

Incubate on ice for 20 minutes

Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.

Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube

Incubate in shaker at 37°C, 225 rpm for 30 min

Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)

Incubate plates at 37°C overnight

Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

DNA Preparations

Materials

DNA miniprrep

LB agar plates, Cat No. Teknova, appropriate antibiotic

DH5a competent cells, Invitrogen, Cat 18265-017

SOC (Recovery Medium), Lucigen, Cat No. F98226

1.5 mL tube

Vortex

Pipet and tips

Ice bucket and ice

Water bath (42°C)

Incubator (37°C) and shaker

Methods

Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells

For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells

Turn on incubator-shaker at 37°C.

Turn on incubator for plates at 37°C.

Set up water bath at 42°C.

Bring to room temperature S.O.C medium.

Bring LB plates supplemented with appropriate antibiotic at room temperature.

Thaw competent cells on ice.

Aliquots competent cells in as many tubes as needed.

Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix

Incubate on ice for 20 minutes

Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.

Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube

Incubate in shaker at 37°C, 225 rpm for 30 min

Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)

Incubate plates at 37°C overnight

Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

Gel

Materials

DNA miniprrep

LB agar plates, Cat No. Teknova, appropriate antibiotic

DH5a competent cells, Invitrogen, Cat 18265-017

SOC (Recovery Medium), Lucigen, Cat No. F98226

1.5 mL tube

Vortex

Pipet and tips

Ice bucket and ice

Water bath (42°C)

Incubator (37°C) and shaker

Methods

Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells

For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells

Turn on incubator-shaker at 37°C.

Turn on incubator for plates at 37°C.

Set up water bath at 42°C.

Bring to room temperature S.O.C medium.

Bring LB plates supplemented with appropriate antibiotic at room temperature.

Thaw competent cells on ice.

Aliquots competent cells in as many tubes as needed.

Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix

Incubate on ice for 20 minutes

Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.

Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube

Incubate in shaker at 37°C, 225 rpm for 30 min

Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)

Incubate plates at 37°C overnight

Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

Gel Purification

Materials

DNA miniprrep

LB agar plates, Cat No. Teknova, appropriate antibiotic

DH5a competent cells, Invitrogen, Cat 18265-017

SOC (Recovery Medium), Lucigen, Cat No. F98226

1.5 mL tube

Vortex

Pipet and tips

Ice bucket and ice

Water bath (42°C)

Incubator (37°C) and shaker

Methods

Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells

For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells

Turn on incubator-shaker at 37°C.

Turn on incubator for plates at 37°C.

Set up water bath at 42°C.

Bring to room temperature S.O.C medium.

Bring LB plates supplemented with appropriate antibiotic at room temperature.

Thaw competent cells on ice.

Aliquots competent cells in as many tubes as needed.

Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix

Incubate on ice for 20 minutes

Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.

Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube

Incubate in shaker at 37°C, 225 rpm for 30 min

Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)

Incubate plates at 37°C overnight

Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

Creating Constructs

Materials

DNA miniprrep

LB agar plates, Cat No. Teknova, appropriate antibiotic

DH5a competent cells, Invitrogen, Cat 18265-017

SOC (Recovery Medium), Lucigen, Cat No. F98226

1.5 mL tube

Vortex

Pipet and tips

Ice bucket and ice

Water bath (42°C)

Incubator (37°C) and shaker

Methods

Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells

For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells

Turn on incubator-shaker at 37°C.

Turn on incubator for plates at 37°C.

Set up water bath at 42°C.

Bring to room temperature S.O.C medium.

Bring LB plates supplemented with appropriate antibiotic at room temperature.

Thaw competent cells on ice.

Aliquots competent cells in as many tubes as needed.

Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix

Incubate on ice for 20 minutes

Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.

Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube

Incubate in shaker at 37°C, 225 rpm for 30 min

Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)

Incubate plates at 37°C overnight

Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

Cloning with pUC Plasmid of Lac Operation (CCA_San_Diego Specific)

Materials

DNA miniprrep

LB agar plates, Cat No. Teknova, appropriate antibiotic

DH5a competent cells, Invitrogen, Cat 18265-017

SOC (Recovery Medium), Lucigen, Cat No. F98226

1.5 mL tube

Vortex

Pipet and tips

Ice bucket and ice

Water bath (42°C)

Incubator (37°C) and shaker

Methods

Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells

For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells

Turn on incubator-shaker at 37°C.

Turn on incubator for plates at 37°C.

Set up water bath at 42°C.

Bring to room temperature S.O.C medium.

Bring LB plates supplemented with appropriate antibiotic at room temperature.

Thaw competent cells on ice.

Aliquots competent cells in as many tubes as needed.

Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix

Incubate on ice for 20 minutes

Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.

Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube

Incubate in shaker at 37°C, 225 rpm for 30 min

Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)

Incubate plates at 37°C overnight

Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

Cloning with Constructive Promoter of different Strengths (CCA_San_Diego Specific)

Materials

DNA miniprrep

LB agar plates, Cat No. Teknova, appropriate antibiotic

DH5a competent cells, Invitrogen, Cat 18265-017

SOC (Recovery Medium), Lucigen, Cat No. F98226

1.5 mL tube

Vortex

Pipet and tips

Ice bucket and ice

Water bath (42°C)

Incubator (37°C) and shaker

Methods

Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells

For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells

Turn on incubator-shaker at 37°C.

Turn on incubator for plates at 37°C.

Set up water bath at 42°C.

Bring to room temperature S.O.C medium.

Bring LB plates supplemented with appropriate antibiotic at room temperature.

Thaw competent cells on ice.

Aliquots competent cells in as many tubes as needed.

Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix

Incubate on ice for 20 minutes

Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.

Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube

Incubate in shaker at 37°C, 225 rpm for 30 min

Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)

Incubate plates at 37°C overnight

Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

Cloning for depository (CCA_San_Diego Specific)

Materials

DNA miniprrep

LB agar plates, Cat No. Teknova, appropriate antibiotic

DH5a competent cells, Invitrogen, Cat 18265-017

SOC (Recovery Medium), Lucigen, Cat No. F98226

1.5 mL tube

Vortex

Pipet and tips

Ice bucket and ice

Water bath (42°C)

Incubator (37°C) and shaker

Methods

Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells

For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells

Turn on incubator-shaker at 37°C.

Turn on incubator for plates at 37°C.

Set up water bath at 42°C.

Bring to room temperature S.O.C medium.

Bring LB plates supplemented with appropriate antibiotic at room temperature.

Thaw competent cells on ice.

Aliquots competent cells in as many tubes as needed.

Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix

Incubate on ice for 20 minutes

Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.

Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube

Incubate in shaker at 37°C, 225 rpm for 30 min

Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)

Incubate plates at 37°C overnight

Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

Clone verification

Materials

DNA miniprrep

LB agar plates, Cat No. Teknova, appropriate antibiotic

DH5a competent cells, Invitrogen, Cat 18265-017

SOC (Recovery Medium), Lucigen, Cat No. F98226

1.5 mL tube

Vortex

Pipet and tips

Ice bucket and ice

Water bath (42°C)

Incubator (37°C) and shaker

Methods

Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells

For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells

Turn on incubator-shaker at 37°C.

Turn on incubator for plates at 37°C.

Set up water bath at 42°C.

Bring to room temperature S.O.C medium.

Bring LB plates supplemented with appropriate antibiotic at room temperature.

Thaw competent cells on ice.

Aliquots competent cells in as many tubes as needed.

Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix

Incubate on ice for 20 minutes

Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.

Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube

Incubate in shaker at 37°C, 225 rpm for 30 min

Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)

Incubate plates at 37°C overnight

Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

Glycerol Stock

Materials

DNA miniprrep

LB agar plates, Cat No. Teknova, appropriate antibiotic

DH5a competent cells, Invitrogen, Cat 18265-017

SOC (Recovery Medium), Lucigen, Cat No. F98226

1.5 mL tube

Vortex

Pipet and tips

Ice bucket and ice

Water bath (42°C)

Incubator (37°C) and shaker

Methods

Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells

For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells

Turn on incubator-shaker at 37°C.

Turn on incubator for plates at 37°C.

Set up water bath at 42°C.

Bring to room temperature S.O.C medium.

Bring LB plates supplemented with appropriate antibiotic at room temperature.

Thaw competent cells on ice.

Aliquots competent cells in as many tubes as needed.

Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix

Incubate on ice for 20 minutes

Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.

Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube

Incubate in shaker at 37°C, 225 rpm for 30 min

Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)

Incubate plates at 37°C overnight

Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

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