Difference between revisions of "Team:Washington/Protocols"

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Polymerase Chain Reaction (PCR)

Overlap Extension PCR

Gibson Assembly

1. Prepare 5x ISO buffer
Components and amounts for ISO Buffer in Gibson Assembly

Component	6mL Reaction
1 M Tris-HCl pH 7.5	3 mL
2 M MgCl₂	150 μL
100 mM dGTP	60 μL
100 mM dATP	60 μL
100 mM dTTP	60 μL
100 mM dCTP	60 μL
1 M DTT	300 μL
PEG-8000	1.5 g
100 mM NAD	300 μL
Water	Add to 6 mL



2. Aliquot 100 μL and store at -20 °C
3. Prepare the Gibson assembly master mixture.
Components and amounts for master mixture in Gibson assembly

Component	1.2 mL Reaction
5x ISO Buffer	320 μL
10 U/μL T5 Exo	0.64 μL
2 U/μL Phusion Pol	20 μL
40 U/μL Taq Ligase	160 μL
Water	add to 1.2 mL



4. Aliquot 15 μL and store at -80 °C
5. Thaw a 15 µL Gibson assembly mixture aliquot and keep on ice until it is ready to be used.
6. Use 7.5 µL of the Gibson assembly mixture. This is enough for one reaction.
7. 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).
8. Incubate at 50 °C for 1 hour. Hold at 4 °C.
9. 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

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.

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)

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).

Yeast Transformation

Components:

• 50% PEG 3350 (240 µL per transformation)
• 1.0 M LiOAc ( 36 µL per transformation)
• Salmon Sperm DNA (boiled) ( 25 µL per transformation)
• Yeast Competent Cell aliquot
• Appropriate LB agar plates

Procedure:

1. Spin down all the Yeast Competent Aliquots on table top centrifuge for 20 seconds
2. Add 240 µL of 50% PEG into each competent aliquot tube.
3. Add 36 µL of 1M LiOAc to each tube
4. Add 25 µL of Salmon Sperm DNA (boiled) to each tube
5. Pipette a volume equivalent to 1ug of plasmid into aliquot tube
6. Vortex each tube on highest settings until the cells are resuspended.
7. Place all aliquots on 42ºC heat block for 15 minutes.
8. Retrieve all tubes from 42ºC heat block.
9. Spin the tube down for 20 seconds on a small tabletop centrifuge.
10. Remove all the supernatant carefully with a 1000 µL pipettor (~400 µL total)
11. Add 200 µL of MG water to the transformed cells
12. Flip the plate(s) and add 4-5 glass beads to it, add 200 µL of mixtures on each plate.
13. Shake the plates in all directions to evenly spread the culture over its surface till dry. Discard the beads in a used beads container. Throw away the 1.5 mL tubes.
14. Incubate all the plates with agar side up at 37ºC

Plate Reader Fluorescence Array

Day 1:

1. Select a colony and inoculate 5mL LB culture with transgenic yeast. Incubate overnight at 220 rpm and 30C.

Day 2:

1. Measure OD of liquid culture and dilute to 10 mL LB final volume with OD 0.05.
2. Grow for 2 hours, until OD 0.4.
3. Place 8 replicates of 100 μL of yeast culture in four rows (32 wells total).
4. Distribute log scale concentrations of our zinc inducer across the 8 wells, ranging from 0 to 100 μM.
5. Take time based assays to measure fluorescence and absorbance over 10 mins, 20 mins, and 30 mins after inducing. These are the settings we will use on the plate reader: