Team:UTK-Knoxville/protocol

PROTOCOL

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Media

Protocol

Experimental Procedures

Timeline

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Chemically Competent cells

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This method is used for E. coli.Chemically Competent cells were used for heat shock transformation:

• Grow seed culture in 6-8mL LB from single colony in 37̊C shaker overnight§ Grow seed culture in 6-8mL LB from single colony in 37̊C shaker overnight
• The next day, subculture seed into a specified volume of SOB medium at a beginning OD of 0.05 and let grow until an OD ~0.5 in 37̊C shaker
• Once cells have reached target OD, dispense into a sterile centrifuge tube and put on ice for 30 minutes
• Centrifuge tube for 10 minutes at 4000rpm and 4̊C
• Discard supernatant and remove remaining supernatant by pipet
• Gently resuspend pellet in 10-30mL ice-cold CCMB80 buffer
• Put on ice for 20 minutes
• Centrifuge for 10 minutes at 4000rpm and 4̊C
• Repeat the last four steps
• Discard supernatant and remove remaining supernatant by pipet
• Resuspend pellet with ice-col CCMB80 to an OD ~3.0
• Aliquot into 1.5mL tubes and store in -80̊C freezer

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Gel Electrophoresis:

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• Add 0.54 g Agarose to 60 mL of TAE buffer (0.8% gel)
• Microwave for 90 seconds
• Let mixture cool down
• Add 6 μL ethidium bromide
• Load the gel with Thermo Scientific GeneRuler 1kb Plus DNA ladder and samples
• Run for 60 minutes at 125V

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Gel Purification:

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For all gel purifications, the ZymocleanTM Gel DNA Recovery Kit (cat. no. D4008) was used.

• Add 3 volumes of ADB to each volume of gel.
• Incubate at 57̊C until fully “dissolved”, removing every 5-10min to vortex sample.
• Load solution into column
• Centrifuge for 30 seconds at 16,000g. Discard the flow through.
• Add 200 μL of DNA wash buffer to the column. Centrifuge for 30 seconds.
• Repeat the wash step.
• Centrifuge for 1 minute at 16,000g to dry out the column
• Check purity via Thermo Scientific nanodrop 2000c spectrophotometer

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Gibson Assembly:

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• 50-100ng backbone DNA
• Incubate at 57̊C until fully “dissolved”, removing every 5-10min to vortex sample.
• __ng insert DNA*
• 15 μL Gibson Assembly Mix
• Sterile DI Water to 20 μL

*ratio of backbone to insert used was 1:1, 1:5, or 1:10 and 1:0 was used as a negative control
Mixture was incubated at 60̊C for 60 minutes, followed by heat shock transformation.

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Heat Shock Transformation:

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All heat shock transformations were done using E. coli.

• Mix 50 μL chemically competent cells with specified amount of plasmid* in pre-chilled, sterile 15 mL tubes. Gently mix by tapping the tube.
• Leave mixture on ice for 30 minutes
• Heat shock in 42̊C water bath for 60 seconds
• Return to ice for 2 minutes.
• Add 950 μL prewarmed SOC medium to the tubes
• Incubate for 40-60 minutes in 37̊C shaker
• Transfer cells to 1.5mL tube and centrifuge at 16,000g for 5 minutes
• Discard supernatant
• Resuspend pellet in remaining supernatant and transfer to prewarmed agar plate**
• Incubate overnight in 37̊C oven

*when doing Gibson Assembly or Ligation, all of the reaction was mixed, when using purified plasmid use 50ng.

**agar plate may contain antibiotic resistance, depending on the plasmid being transformed into the cells.

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Ligation

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For all ligation reactions, T4 ligase and buffer were used. The recipe is as follows:

• 100ng backbone DNA
• __ng insert DNA*
• 1 μL T4 Buffer
• 1 μL T4 DNA Ligase
• Sterile DI Water to 10 μL

*ratio of backbone to insert used was 1:1, 1:5, or 1:10 and 1:0 was used as a negative control
Mixture was incubated at room temperature for 30 minutes, followed by heat shock transformation.

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PCR Cleanup/Gel Cleanup Ligation

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This method was used when pieces from the gel purification step were not as pure as desired or if a small amount of PCR reaction was loaded into the gel and the rest was in need of purification.

For all PCR Cleanup/Gel Cleanup, the DNA Clean & ConcentratorTM -5 (cat. no. D4014) kit was used.

• Add 2-7 volumes of DNA Binding Buffer to each volume of DNA sample
• For plasmid or genomic DNA >2 kb, add 2 volumes
• For PCR or short DNA fragments, add 5 volumes
• For ssDNA purification, add 7 volumes
• Load the mixture into a Zymo-Spin Column in a Collection Tube
• Centrifuge at 16,000g for 30 seconds. Discard the flow through
• Add 200 μL of DNA Wash Buffer to the column and centrifuge for 30 seconds. Repeat the wash step.
• Place the Zymo-Spin Column into a new 1.5 mL tube. Add 10-15 μL of DNA Elution Buffer or Sterile DI Water directly to the column matrix
• Let stand for ~5 minutes
• Centrifuge at 16,000g for 1 minute to elute the DNA
• Check purity via Thermo Scientific nanodrop 2000c spectrophotometer

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PCR Reaction (phusion)

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This type of PCR reaction used Thermo Scientific Phusion Hot Start II High-Fidelity DNA Polymerase, Thermo Scientific dNTP Mix (2mM), Thermo Scientific 100% DMSO, and Thermo Scientific 5X Phusion HF Buffer. The phusion reaction was used for amplifying pieces of DNA for plasmid construction. The following recipe was used:

• 1pg-10ng template DNA
• 5 μL dNTPs
• 10 μL Buffer
• 1.5 μL DMSO
• 0.5 μL phusion polymerase
• 0.5 μL each of primers
• Sterile DI Water to a final volume of 50 μL

Cycles for the PCR were laid out as such:

STEP	TEMP	TIME
Initial Denaturation	94°C	5 minutes
Denaturation	94°C	5-10 seconds
Annealing	*50–72°C	10-30 seconds
Extension	72°C	15-30 seconds/kb
Final Extension	72°C	10 minutes
Hold	4–10°C

PCR Was run for 25-35 cycles.

*Temperature determined by primer melting temperature

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PCR Reaction (Taq)

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This PCR used Lucigen EconoTaq PLUS Green 2X Master Mix, which contains everything needed for a PCR except template and primers. This reaction was used mainly for colony PCR, in which a colony was picked from a plate and resuspended in 5 μL of medium or sterile water. 2 μL were used as template for the PCR while the rest was transferred to appropriate medium and grown. The following recipe was used:

• 2 μL template DNA
• 0.5 μL each of primers
• 12.5 μL Taq polymerase Master Mix
• Sterile DI Water to a final volume of 25 μL

Cycles for the PCR were laid out as such:

STEP	TEMP	TIME
Initial Denaturation	94°C	5 minutes
Denaturation	94°C	5-10 seconds
Annealing	*50–72°C	10-30 seconds
Extension	72°C	1 minute/kb
Final Extension	72°C	10 minutes
Hold	4–10°C

PCR Was run for 25-35 cycles.

*Temperature determined by primer melting temperature

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Plasmid Extraction:

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For all plasmid extractions, the ZR Plasmid MiniprepTM-Classic (cat. no. D4016) kit was used.

• Pellet 0.5-5 mL overnight culture. Discard the supernatant.
• Add 200 μL of P1 Buffer (red). Resuspend the pellet. Transfer to a 2.0 mL tube.
• Add 200 μL of P2 Buffer (green). Gently mix by tapping/flicking. Incubate at room temperature for 1-3 minutes.
• Add 400 μL of P3 Buffer (yellow). Mix thoroughly.
• Centrifuge at 16,000g for 10 minutes.
• Load the supernatant into a column.
• Centrifuge for 30 seconds.
• Discard the flow through.
• Add 200 μL of Endo-wash buffer. Centrifuge for 15 seconds.
• Add 400 μL of Plasmid wash buffer. Centrifuge 30 seconds.
• Centrifuge for 1 minute to dry residual wash buffer from column.
• Place the column in a 1.5 mL tube. Add 50 μL of sterile (autoclaved) DI water. Let stand for 5 minutes.
• Centrifuge for 30 seconds.
• Check purity of sample via Thermo Scientific nanodrop 2000c spectrophotometer

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Plating Cells

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• Use plate with the appropriate resistance for each sample.
• Transfer culture to a 1.5 mL or 2.0 mL tube and centrifuge at 16,000g for 5 minutes
• Decant liquid and resuspend pellet in remaining liquid.
• Pipet concentrated cell mixture onto plate.
• Add 15-20 sterile glass beads to plate.
• Cover the plate and roll the beads around the surface.
• Incubate at 37̊C.

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Preparation of -80̊C Stock

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• Grow cells to mid log phase.
• Label 2 mL cryotubes with the strain, plasmid, antibiotic resistance, initials, and the date.
• Pipet 750 μL of 30% glycerol into the tubes.
• Add 750 μL of mid log phase culture.
• Mix well and store in the appropriate box.
• Update the database.

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Restriction Digest

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Restriction digest mixing was performed on ice using the following recipe and were incubated in a 37̊C water bath for 2-16 hours.

• 5 units restriction enzyme
• 500 ng of DNA template
• 2.5 μL of 10x NEBuffer or NEB Cutsmart buffer
• 0.5 μL of 100x BSA, no BSA when using NEB Cutsmart buffer
• Sterile DI water
• Total volume: 25μL