Team:Tec-Monterrey/Protocols

1. Plasmid DNA Extraction
2. Restriction Digestion
3. Agarose Gel Electrophoresis
4. Purification of DNA Fragments from an Agarose Gel
5. DNA Ligation
6. Preparation of Electrocompetent Cells
7. Transformation via Electroporation

Plasmid DNA Extraction

Wizard® Plus SV Minipreps DNA Purification System was used for DNA extraction. Slight changes were made to the Centrifugation protocol:

Production of Cleared Lysate

1. Pellet 20 mL of overnight culture for 15 minutes at 8,000 x g.

12. Centrifuge for 2 minutes at 14,000 x rpm with the microcentrifuge lid open to allow evaporation of any residual ethanol.

14. Add 50 μL of Nuclease-Free Water to the Spin Column. Centrifuge at 14,000 rpm for 1 minute at room temperature.

Restriction Digestion

1. Review the Digestion Calculator, available through here, to make the proper calculations. Adjust the final volume of the reaction and be sure to add the DNA concentration and the proper enzymes to be used (with their corresponding quantities).
2. Add all of the components but the enzymes to a 0.2 ml PCR tube. Make sure that the buffer used is the one recommended for the combination of enzymes to be used.
3. Carefully, add the enzymes, preferably without taking them out of the freezer.
4. Incubate the tubes at 37˚C for two hours.

Agarose Gel Electrophoresis

The following procedure is for the preparation of a 1% agarose gel.

Materials:

• Agarose (Electrophoresis grade)
• 1X TAE
• Electrophoresis chamber
• Electrophoresis tray
• Transilluminator
• SYBR Safe
• Microwave
• Beaker

Procedure:

1. Take a beaker and place 30 mL of 1X TAE.
2. Weigh 0.3 gr of agarose and add it to 1X TAE solution.
3. Place beaker inside microwave and heat it until agarose dissolves completely.
4. Allow agarose to cool down to 60°C and add 3 μL of SYBR Safe 10 000X.
5. Mix solution and pour it in a taped gel tray.
6. Add a comb and let the gel harden for about 15 minutes.
7. Remove the comb and the tape and put the gel tray in the electrophoresis chamber.
8. Add enough 1X TAE to completely cover the gel.
9. Add DNA loading buffer to the samples and load them.
10. Run the gel at a voltage between 60V and 120V, depending on the desired resolution/time.
11. Visualize or cut the DNA by putting it in the transilluminator.

Purification of DNA Fragments from an Agarose Gel

Wizard® SV Gel and PCR Clean-Up System was used for purification of DNA fragments obtained from an agarose gel or ligation reaction. Slight changes were made to the DNA Purification by Centrifugation protocol:

Washing

6. Empty the Collection Tube, incubate at room temperature for 5 minutes and recentrifuge the column assembly for 2 minutes at 8,000 x rpm to allow evaporation of any residual ethanol.

8. Add 30 μL of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute. Centrifuge at 16,000 x g for 3 minutes.

DNA Ligation

T4 DNA Ligase protocol by Invitrogen was used to ligate DNA, aided by a Ligation Calculator available through here.

Preparation of Electrocompetent Cells

The following protocol is for preparing electrocompetent E.coli cells.

• LB-agar plate
• LB medium
• 500 mL chilled autoclaved dH2O, stored at 4°C
• Chilled 10% glycerol/dH2O solution, stored at 4°C
• 1 L flask
• 50 ml tubes
• 0.2 mL tubes
• Ice bucket
• Ice
• Refrigerated centrifuge

Procedure:

1. Streak Escherichia coli DH5α cells onto LB-agar plate with no antibiotics and incubate at 37°C overnight.
2. Pick one colony and place it in a 50 mL tube with 20 mL LB medium. Incubate overnight on a shaker at 37°C and 350 rpm.
3. Add 250 ml of LB medium to a flask and add the overnight culture until an OD600 of 0.1 is reached.
4. Place the flask on a shaker at 37°C, 350 rpm until an OD600 between 0.4-0.6 is reached.
5. Transfer the diluted culture to 50 mL tubes.
6. After this step, the cells must be kept at 4°C at all times. Place the cells on ice for 15 minutes.
7. Cool the centrifuge to 4°C.
8. Centrifuge the tubes for 10 min at 8000g at 4°C.
9. Remove supernatant and gently resuspend pellets with 10 mL cold sterile water by pipetting. Add the rest of the water to a total volume of 50 mL.
10. Centrifuge a second time for 10 min at 8000g at 4°C.
11. Remove supernatant and gently resuspend pellets with 10 mL cold sterile water by pipetting. Add the rest of the water to a total volume of 50 mL.
12. Centrifuge a third time for 10 min at 8000g rpm at 4°C.
13. Remove supernatant and gently resuspend pellets with the remaining water (if it’s too little, add some more).
14. Calculate and add glycerol so that the final concentration is 10-15 %.
15. Resuspend the cells and aliquot 50 μL per 0.2 mL tube (tubes on ice) and store at -80°C.

Transformation via Electroporation

The following protocol is for transforming plasmid DNA into Escherichia coli DH5α cells.

• Electrocompetent cells
• Plasmid DNA (from a ligation reaction)
• Ice
• Ice bucket
• Electroporation cuvette (2mm gap width)
• Electroporator (Biorad Gene Pulser Xcell)
• LB-agar plate with appropriate antibiotic
• 1mL SOC at room-temperature

Procedure:

1. Chill electroporation cuvettes, DNA samples and tubes on ice.
2. Once cuvettes are cold, remove electrocompetent cells from -80°C freezer and thaw on ice.
3. Turn on electroporator and set voltage to 2.5 kV.
4. Dial a micropipette to 1 or 2μL of DNA sample.
5. Dial a micropipette to 50μL of electrocompetent cells.
6. Dial a micropipette to 1000μL and pipet in SOC. Place micropipette on counter such that tip doesn't touch anything.
7. Pipet 1-2μL of DNA sample and place inside the cuvette.
8. Pipet 50μL of electrocompetent cells inside the cuvette ensuring they mix with the DNA sample. Do not pipet up and down.
9. Place cuvette back on ice to ensure it remains cold.
10. Tap the cuvette on the counter gently so that cells are at the bottom and to remove any air bubbles.
11. Wipe off excess moisture from outside of cuvette.
12. Place in chamber of electroporator so that the cuvette sits between electrodes.
13. Pulse the cells with a shock by pressing button on electroporator.
14. Remove cuvette from the chamber and immediately add SOC.
15. Transfer cuvette to 37°C incubator and shake at 350 rpm to promote aeration. Incubate for 1 hr.
16. Plate 100 μL transformation onto LB-agar plate supplemented with appropriate antibiotic.
17. Incubate plate overnight at 37°C until colonies appear.