Team:UTK-Knoxville/protocol

From 2014.igem.org

PROTOCOL

Media

Protocol

Experimental Procedures

Timeline

Chemically Competent cells

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

Gel Electrophoresis:

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

Gel Purification:

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

Gibson Assembly:

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.

Heat Shock Transformation:

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.

Ligation

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.

PCR Cleanup/Gel Cleanup Ligation

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

PCR Reaction (phusion)

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

PCR Reaction (Taq)

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

Plasmid Extraction:

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

Plating Cells

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.

Preparation of -80̊C Stock

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.

Restriction Digest

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