<"font color=blue">Separating Gel

1) Level glass plates in the casting frame and place the frame within the casting stand.Ensure the plates are locked into place.
2) Place the following solutions in a 50ml falcon tube:

Mix:

- 4.2ml DH20
- 2.5ml TRIS 4X Buffer.
- 3.3ml Polyacrylamide Solution.

3) Mix and invert six times
4) Add 33.3μl of APS (Ammonium Persulfate) into the falcon tube.
5) Mix and invert six times
6)Add 6.7μl of TEMED to the falcon tube.
NOTE: Must be done in a fumehood. TEMED is toxic.

7) Mix and invert six times. Solution will harden in roughly 15 minutes.
8) Add solution in between glass plates using a pipette aid roughly 3/4 the way up the front plate.
9) Fill remaining space with ethanol to ensure an even leveling of the gel.
10) Once gel is solidified, remove the ethanol using a kim wipe ensuring the are in bewteen the plates is now dry.

Stacking Gel:

1) Place the following solutions in a 50ml falcon tube:

Mix:

- 3.05ml DH20
- 1.25ml Stacking Buffer Solution
- 650μl Polyacrylamide Solution.

2) Mix and invert six times.
3) Add 25μl of APS ( Ammonium Persulfate ) into the falcon tube.
4) Mix and invert six times.
5) Add 5μl of TEMED to solution.
NOTE: Must be done in a fumehood. TEMED is toxic.

6) Mix and invert six times.
7) Fill in the remaining area in between the glass plates with the stacking solution and place comb inside.
8) After gel is solidfied, remove from casting frame and place in plastic wrap. Store in the fridge.
READY FRO USE!

Electrophoresis

1) Prepare samples that will be ran in the gel electrophoresis.
2) Mix 15μl of each sample with 5μl of sample buffer solutiom. A 3 to 1 ratio is used. A maximum of 30μl can be inserted into each well.
3) Place glass plates in the electrode assembly and into the tank cell.
4) Fill tank with 1X electro buffer solution. Using a loading pipette and tips, load desired samples into wells along with a molecular weight ladder sample.
5) Connect the lid to the tank and place the correct cables onto the tank lid. Run gel at 120 volts for 60-90 minutes or until desired bands are about the length of the gel.

1) Once SDS-PAGE is complete, remove gels from the glass plates and remove the stacking gel layer using a paper towel.
2) Place gel in a glass container, wash with 200ml distilled water for 5 minutes. Repeat the wash step 4X. (shake gel back and forth in shaker) Decant water into waste container.
3) Mix a solution with 90ml of 10% acetic acid and 90ml of 10% methanol in a clean beaker. Add 10ml of fixative enhancer solution into the beaker and pour over the gel. Let sit for 20 minutes on shaker, or up to a maximum of overnight, depending on the desired sensitivity of the gel.
4) Decant solution into a proper waste container.
5) Wash gel once again with 200ml of distilled water for 5 minutes on shaker. Repeat 4X. Decant water into waste container.
6) Mix both parts of stain solution into two separate falcon tubes.

Mix 1

- 2.5ml of Silver Complex Solution
- 2.5ml of Reduction Moderator Solution
- 2.5ml of Image Development Reagent
- 17.5ml of DH20

Mix 2

- 1.25g of Development Accelerator
- 25ml of DH20

7) Mix both solutions together until each are dissolved, pour over gel. Let sit for up to 20 minutes or until desired bands show.
8) Decant solution into proper waste container.
9) Prepare a stop solution containing 90ml of 10% acetic acid and 90ml of 10% methanol. Pour over gel and shake for 15-20 minutes.
10) Decant solution into proper waste container.
11) Wash gel with 200ml of DH20 for 5 minutes on shaker. Repeat 2X.
GELS ARE STAINED!

Ordering with IDT was tricky because we could not order sequences that were highly repetitive. Unfortunately, the unique characteristics we found in the colour producing proteins in literature involved high concentrations of specific amino acids.

To overcome this limitation, we had to insert amino acids that we believed had no colour in between the amino acid of choice.

Before beginning, ensure all reaction components and properly thawed and mixed.

Calculate the required volumes of each component based on the following table:

- Reaction Volumes may be adjusted between 10 - 50 µL
- < 1 ng less complex DNA (0.1 to 1.0 ng) per 50 ╡L

1. Transfer the appropriate volumes of PCR master mix template and primer to individual PCR tubes
2. Cap or seal individual reactions.
3. Mix and centrifuge briefly.

NOTE:A PCR gradient was conducted to determine the optimal annealing temperature needed for best results. We observed that the best annealing temperature was 61°C.

1. Perform PCR with the following Cycle Protocol

NOTE: PCR products can be left overnight at 4°C

1. Add 5X Buffer PB to 1X of the PCR reaction and mix into a separate 1.5 mL microcentrifuge tube. If the colour of the mixture is orange or violet, add 10 µK 3M sodium acetate, pH 5.0 and mix. The colour of the mixture will turn yellow
2. Place a QIAquick column into a provided 2 mL collection tube and then place into centrifuge and transfer mix
3. To bind DNA, apply the samples to the QIAquick colum and centrifuge at 13 000 rpm from 60s. Discard the fluid that is in the collection tube (aka the flow-through). Place column back into the same tube.
4. Centrifuge the column once more in the provided 2 mL collection tube for 1 minute to remove residual wash buffer
5. Dab the bottom of the column on Kim Wipe tissue.
6. Place each column in a clean 1.5 mL microcentrifuge tube
7. Add 50 µL of EB to the column (Aim for the center of the membrane inside the column)
8. Centrifuge at 13 000 RPM for 60 s
9. DO NOT THROW OUT FLOW THROUGH. Using a micropipette, set at 65 µL, take flow through and add it once more to the column
10. Centrifuge again (13 000 RPM, 60s)
11. Dispose of column (Purified DNA should now be in the 1.5 mL microfuge tube)