实验步骤

1. Preparing the Glass plates

1) Locate two LiCor plates (one with a notch and one without) and clean them as follows:

a. Scrape off the polyacrylamide with a razor blade

b. Scrub both sides of the plate with detergent and water using crub brush and wet paper towel.

c. Rinse the plate well with tap water, running your hands over the gel to detect any residual polyacrylamide.

d. Set the plates on the drying rack to drain.

e. Dry the plates completely using kimwipes (not paper towels)

f. Clean the plates scrupulously with di-water and kimwipes. Be super-scrupulous with the side of each plate which will contact the polyacrylamide.

2) Assemble the plates using 0.25 mm spacers on each side. Set it up so that the plate without the notch is on the bottom and the notched plate is on top with the notch facing you. Place the clamps on and add the U-shaped plexiglass spacer to the slot in the top and snug down the clamps (firm but not tight). Pick up the assembly and admire your work to see that everything is lined up correctly.

2. Preparing the Gel mix.

1) Make the gel mix in a 50 mL polypropylene tube.

Add: 10.5 g urea (ultrapure)

3.0 mL of Long Ranger Acrylamide Mix (50% Acrylamide)

3.0 mL of 10x TBE buffer

ddH2 O to 25 mL

Mix until the urea is completely dissolved.

2) Filter the gel mix using a 0.22 um pore filter unit and a 30 mL syringe.

3) Degas the gel mix. - Transfer the gel mix to a small side arm flask and place a stopper in the top. Attach the side arm to a vacuum source and apply vacuum to the flask while agitating for 1-2 min. Continue the application of a vacuum until bubbles no longer appear when flask is shaken (1-2 minutes).

4) Place the flask on ice.

3. Pouring the gel

1) Gather the following items: Pipettor set to 100 uL, pipettor set to 10 uL, 10% ammonium persulfate solution (APS: stored in refrigerator and less than a week old), TEMED, a 30 mL syringe, and the spacer for the top of the gel.

2) Pull the stopper out of the syringe, fill the pipettors with 100 uL APS and 10 uL TEMED.

3) Add 100 uL APS and 10 uL TEMED to the gel mix, agitate gently to mix without introducing bubbles. While holding your finger over the end of the syringe pour the gel mix into the syringe and put the plunger just into the barrel of the syringe. Invert the syringe and use the plunger to expell the air.

4) Pour the gel, being careful not to introduce bubbles (bang on the plates while pouring).

5) Insert the top spacer and place three clamps on the top of the gel. 6. Allow 45 min for polymerization.

4. Pre-electrophoresis

1) Carefully pull the spacer out and use a razor and wet kimwipes to remove excess polyacrylamide and dried urea from the top of the gel.

2) Make sure the outsides of the gel plates are clean and dry, especially in the area where the scanner will be pointing.

3) Assemble the gel rig on the sequencer, being careful not to get any liquid behind the glass plates.

4) Make 1000 mL of 0.6x TBE buffer and fill the buffer chambers.

5) Use the syringe with the bent needle to get rid of bubbles clinging to the bottom of the plates.

6) Attach the top and bottom buffer chamber covers, attach the cable between the top buffer chamber and the sequencer, and close the sequencer.

7) Run the gel for 45 minutes.

5. Denature the sequencing reactions and prepare them for loading.

1) Place the reactions in a heat block at 90℃ for 5 minutes and cool them quickly on ice.

2) Transfer 10 uL of the reaction from underneath the oil (wipe the oil off the tip) to a clean, labelled tube.

3) Store on ice in dark until ready to load.

6. Loading and running the gel

1) Turn off the power to the gel and the scanner. Open the sequencer and remove the top of the upper buffer chamber.

2) Use a pasteur pipet to flush out the urea which has diffused from the gel into the top well.

3) Insert the sharkstooth comb so that the teeth rest firmly on the gel, but are not digging into the top of the gel.

4) Load the gel (1.2 - 1.5 uL per lane) by placing the end of the pipet tip in the space between the glass plates, directly over the well. Dispense the contents of the tip slowly. You should be able to see the contents run down into the bottom of the well.

5) After all the wells have been loaded, put the top back on the upper chamber, close the sequencer, and start the gel running. Let it run for 10 minutes and set the autogain and focus.