Team:Washington/Protocols

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Protocols: Experiments

Polymerase Chain Reaction (PCR)

Table 1. Components and amounts for PCR.

Table 2. Thermocycling conditions for PCR.

Overlap Extension PCR

You need to make up two separate master mixes called A and B and them store on ice. Place Master Mix A in the thermal cycler first.

Table 3. Components and amounts for Overlap Extension PCR.

Table 4. Thermocycling conditions for Overlap Extension PCR.

Gradient PCR

Table 5. Components and amounts for Gradient PCR.

Table 6. Thermocycling conditions for Gradient PCR.

Touchdown PCR

Table 7. Components and amounts for Touchdown PCR.

Table 8. Thermocycling conditions for Touchdown PCR.

Gibson Assembly

1. Prepare 5x ISO buffer.


Table 9. Components and amounts for ISO Buffer in Gibson Assembly.

2. Prepare the Gibson assembly master mixture.


Table 10. Components and amounts for master mixture in Gibson assembly.

3. Thaw a 15 µL Gibson assembly mixture aliquot and keep on ice until it is ready to be used.
4. Use 7.5 µL of the Gibson assembly mixture. This is enough for one reaction.
5. Add 2.5 µL of DNA to be assembled to the master mixture. The DNA should be in equimolar amounts. Use ~50 ng of each ~6 kb DNA fragment. For larger DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 125 ng of each 15 kb DNA segment).
6. Incubate at 50 °C for 1 hour. Hold at 4 °C.
7. Either immediately freeze at -20 °C or transform 1-2 µL of the assembly reaction into 30-50 µL electro-competent E.coli.

Gel Electrophoresis

Table 11. Agarose gel formulations and DNA separations.

Pouring a Standard 1% Gel

1. Measure 1 g of agarose.
2. Pour agarose powder into microwavable flask along with 1x TAE. Use ~65 mL for purification and ~35 mL for diagnosis.
3. Microwave for 1-3 minutes until the agarose is completely dissolved with sufficient bubbling.
4. Let agarose solution cool down for 5 minutes.
5. Add SYBR Safe stain to the molten agar and swirl to mix. The stain binds to the DNA and allows you to view the DNA bands under an ultraviolet blue light.
6. Pour the agarose into the gel tray with the well comb in place. Use the thick side of the comb if you are purifying from the gel.
7. Place newly poured gel at 4 °C for 10-15 minutes or let it sit at room temperature for 20-30 minutes until it has completely solidified.

Loading Samples and Running an Agarose Gel

1. Add loading buffer (dyed) to each of the digest samples.
2. Once solidified, place the agarose gel into the gel box (electrophoresis unit).
3. Fill gel box with 1x TAE (or TBE) until the gel is covered.
4. Carefully load a molecular weight ladder into the first lane of the gel and your samples in the additional wells of the gel.
5. Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel.
6. Turn off power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
7. Use any device that has blue light to visualize your DNA fragments.

Chemical Transformation

1. Take chemically competent cells (CC) out of -80 °C and thaw on ice.
2. Take agar plates (containing the appropriate antibiotic) out of 4 °C to warm up to room temperature or place in 37 °C incubator.
3. Mix 1 to 5 µL of DNA (usually 10 pg to 100 ng) into 20-50 µL of competent cells in a microcentrifuge or falcon tube. Gently mix by flicking the bottom of the tube with your finger a few times.
4. Place the competent cell/ DNA mixture on ice for 20-30 minutes.
5. Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42 °C water bath for 30-60 seconds (45 seconds is usually ideal, but this varies depending on the competent cells you are using).
6. Put the tubes back on ice for 2 minutes.
7. Add 200-500 µL LB or SOC media (without antibiotic) and grow in 37 °C shaking incubator for 45 minutes.
8. Plate some or all of the transformation onto a 10 cm LB agar plate containing the appropriate antibiotic.
9. Incubate plates at 37 °C overnight.

Electrical Transformation

1. Thaw SOC or LB medium.
2. Thaw E.coli electro-competent cells on ice.
3. Add 0.5-1 µL of plasmid DNA sample to a sterile 1.5 mL tube (don't forget a negative control). Volume should not be over 4 µL. Store on ice.
4. If necessary, sterilize electroporation cuvettes (1 mm gap for E.coli) by rinsing with EtOH, and treating with UV for 2-5 minutes. Store cuvettes on ice.
5. Set the Bio-Rad Gene Pulser to 1.8 kV by pressing the up and down arrows simultaneously. All other settings (200 ohms, 25 µF) will always remain the same.
6. Once cells are thawed, add 30-50 µL cells per tube of DNA. Mix gently 2 times up and down with a pipette tip.
7. Add the cells and DNA directly to the cuvette. To avoid air bubbles, gently tap the cuvette on the bench top 2 or 3 times. You don’t have to put the cap back on.
8. Wipe off the metal electrodes of the cuvette with a kimwipe to remove any water.
9. Place the cuvette in the sample chamber. Be sure that the electrodes are contacting both sides of the chamber.
10. Press and hold both red "pulse" buttons at the same time to deliver the electrical pulse. You will hear a beep once the pulse has been delivered within 1 or 2 seconds. Once you hear the beep, release the buttons. Most readings will be around 4-6 msec following the pulse.
11. If you hear and see a loud pop, the sample has arced (meaning the pulse did not pass through the cells, but somehow around it). Discard the cuvette and try the sample again.
12. Remove the cuvette from the chamber and add 200 µL SOC or LB to resuspend the cells. Add 200 µL of the cells and media to a round bottom snap-cap tube.
13. Incubate at 37 °C for 1 hour.
14. Plate all the cells on LB + antibiotic using glass beads.
15. Grow overnight at 37 °C.

Miniprep

1. Grow 1-5 mL plasmid-containing bacterial cells in LB medium with appropriate antibiotics overnight.
2. Pellet the cells by centrifuging for 1-2 minutes. Decant the supernatant and remove all medium residue by pipet.
3. Completely resuspend the cell pellet in 200 µL of MX1 Buffer by vortexing or scratching the bottom of the tube against a tube rack.
4. Add 250 µL of MX1 Buffer by vortexing or scratching the bottom of the tube against the tube rack.
5. Add 350 µL MX3 Buffer to neutralize the lysate and gently mix the solution immediately.
6. Centrifuge for 5-10 minutes.
7. Transfer the supernatant to a GenCatch plus Column/Collection Tube.
8. Centrifuge for 30-60 seconds at 5000 rpm. Discard flow through.
9. Wash the column once with 500 µL WN buffer by centrifuging for 30-60 seconds at 7000x g (9000 rpm) Discard flow through.
10. Wash the column once with 700 µL WS Buffer by centrifuging for 30 seconds at 7000x g (9000 rpm). Discard flow through.
11. Centrifuge the column at 10,000x g (13000 rpm) for another 2 minutes to remove residual ethanol.
12. Place column into a new 1.5 mL centrifuge tube. Add 50 µL of elution buffer onto the center of the membrane.
13. Stand the column for 2 minutes at room temperature and centrifuge for 30 seconds at 13000 rpm and elute DNA.
14. Store plasmid DNA at 4 °C or -20 °C.

Smash and Grab (Yeast Genomic Prep)

Table 12. Components and amounts for Smash and Grab buffer.

1. Grow 3 mL yeast cultures in selective media overnight in the shaker at 30 °C.
2. Cool 70% EtOH in a -20 °C freezer.
3. Collect cells by centrifugation in a 1.5 mL tube for 1 minute at 2500 rpm, then remove supernatant and repeat until the entire culture is pelleted.
4. Add 1 mL sterile H₂O to each pellet. Vortex and collect cells by centrifugation (same as above).
5. Remove supernatant with a vacuum aspirator and add 200 µL of Smash and Grab Buffer, 0.8 mm glass beads (enough so that the surface of glass beads reaches ~0.2 mL mark on the tube, NOT the liquid) and 200 µL of phenol/chloroform/iso-amyl alcohol (or isopropanol) (kept at 4 °C to each cell pellet (The top layer in the bottle is alcohol, make sure to take phenol/chloroform from deep under the surface). Add the phenol/chloroform inside the hood and be sure the caps are closed tightly.
6. Vortex for 3 minutes (using the multi-tube adaptor on the vortex genie in the hood, at maximum speed)
7. Centrifuge at room temperature for 7 minutes at 14,000 rpm.
8. Remove the upper aqueous layer (about 180 µL) and transfer to a fresh microfuge tube. Discard tube with phenol/Chloroform in the phenol/chloroform waste collection jug.
9. Add 126 µL of room temperature isopropanol (0.7 volume; or 70% of total volume of aqueous layer collected) and invert 4-6 times to mix.
10. Leave at room temperature for 5 minutes.
11. Centrifuge at room temperature for 10 minutes at 14,000 rpm.
12. Remove supernatant with the vacuum and wash pellets of DNA with 200 µL of COLD 70% ethanol. Do not try to resuspend the pellet. Vortex briefly to wash sides of tube with the ethanol.
13. Remove as much ethanol as you can with the vacuum aspirator.
14. Allow pellets to air dry 10-20 minutes at room temperature (the pellets will turn transparent with a greenish tint).
15. Add 50 to 100 µL TE pH 8 (depending on pellet size).
16. Add 0.25 µL RNase A (20mg/ml stock found at 4 °C)
17. Pipet gently to mix (pellet may need to sit in TE for a few minutes to easily resuspend).

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