PREPARATION OF SEQUENCING GELS

MATERIALS:

2-glass plates

1 sharks -tooth comb and spacers

Whatman 3 mm paper

30 or 40% acrylamide-bis (19:1)

10X TBE

urea

10% ammonium persulfate

TEMED

60 cc. syringe

Rain-ex

Preparation of glass plates:

Wash and rinse well the sequencing plates. Silanize the smaller notched plate as follows. Apply a small amount of Rain-ex to the inside surface of the small plate with a Kimwipe. Let air dry to a thin film. Rinse well with distilled water and dry. An additional cleaning with a glass cleaner is helpful. Clean and dry the thermoplate and place over benchwrap paper, elevated but level. Lay spacers along the sides even with the top and bottom of the plate. Place notched plate, silanized- side down, on top of larger plate and clamp.

Preparation of gel solution for 100 ml of an 6% 8 M urea gel:

15 ml of 40% acrylamide-bis (19:1)

10 ml of 10X TBE

46 g urea

~30 ml water

Place in a beaker with a stir bar and place this beaker inside a larger beaker containing some warm water. Stir until dissolved. Do not heat. Measure volume and add water to 100 ml. Add 666 μl 10% ammonium persulfate and 40 μl TEMED and stir gently. Bring up solution in a 60 cc. syringe and let solution flow down center of notched plate being careful not to generate any bubbles. When plates are filled, place flat-edge of shark tooth comb in a few mm - not all the way. Clamp the top of the gel to hold the comb and allow the gel to polymerize (1-2 hr). Gels can be prepared in advance if the top of the gel is wrapped in Saran Wrap.

RUNNING SEQUENCING GELS

To run, set-up the gel in the electrophoresis chamber before removing the comb. Add 1X TBE buffer to the upper chamber and gently remove comb. Immediately rinse unpolymerized acrylamide and urea from the comb area. Repeat this again before you load your samples. Add buffer to the bottom chamber only when you are confident that the upper chamber is not leaking. Prerun the gel 35-50 watts for 30-60 min. The gel should be warm to the touch and the samples should be loaded onto a warm gel. To load samples, turn off power, rinse comb area, and place sharks tooth combs such that the tip of the comb just meets the tip of the gel. Load preheated samples between the tips. If a number of templates are being run, you can avoid too much diffusion of your samples into adjacent lanes by running the DNA for 1 or 2 samples into the gel before loading the next set. Run the gel at 35-40 watts until the bromophenol blue gets close to the bottom. To double load, add additional sample at this time and run the gel until the bromophenol blue gets close to the bottom. Disassemble the plates carefully making sure the gel sticks to the bottom plate. Add fixative solution to gel and let sit for >15 min. Drain excess fixative solution off of the gel and place a piece of Whatman paper on the gel and peel off. Dry the gel thoroughly on a gel dryer with Saran Wrap on top of gel only. Dry for 30-90 min at 80EC. Remove Saran Wrap before exposing the film. (If, by chance, the film should stick to the gel when it is time to expose it, DO NOT PUT THE FILM IN THE PROCESSOR. NOTIFY THE INSTRUCTOR). (For 32 P gels, a Saran- Wrapped-wet gel can be exposed to film without fixing or drying). For 35 S gels, expose for 20-24 hr.