Wash 2 glass plates with Alconox detergent, then rinse and dry.

Clean glass plates with 95% ethanol using a Kimwipe.

Wash 1 comb and 2 spacers with Alconox detergent, then rinse and dry.

Assemble glass plate sandwich according to directions:

a. First place the two spacers on the ends of the large glass plate and then set the smaller glass plate on top.

b. Using the two clamps with the arrows facing in, clamp the sandwich loosely together.

c. Put the loosely clamped sandwich into the gel stand. Loosen the two clamps and put the spacer between the two glass plates. Pushing in on the sides of both the clamps, tighten the clamps.

d. Then loosen one clamp, push down on the spacer and glass plates so that they are aligned and re-tighten the clamp. Repeat for the other side.

e. Take sandwich out and check to see if the two glass plates and spacer are smooth by running your fingernail over them.

f. Then put the glass plate sandwich on the stand and tighten down.

Prepare resolving gel (36mls for 1 gel and 72mls for 2 gels):

One Gel: Two Gels:

g. 14.5mls 1M Tris, pH 8.8 29mls 1M Tris, pH 8.8

h. 9.6mls 30% Acryl 19.2mls 30% Acryl

i. 11.6mls H2O 23.2mls H2O

j. 180ul 20% SDS 360ul 20% SDS

k. 100ul 10% APS 200ul 10% APS

l. 50ul TEMED 100ul TEMED

Add APS and TEMED to resolving gel solution last.

Put in resolving gel using 10ml pipette about 2 inches below the bottom plate.

Add 1ml H2O drop-wise to the top of resolving gel.

Tilt gel to make even.

Put the remaining resolving gel in 15ml conical tube so that you know when gel has polymerized.

Prepare samples and Marker.

Prepare stacking solution (15mls for 1 gel and 30mls for 2 gels):

One Gel: Two Gels:

m. 2ml 30% Acryl 4mls 30% Acryl

n. 1.9mls 1M Tris, pH 6.8 3.8mls 1M Tris, pH 6.8

o. 11mls H2O 22mls H2O

p. 75ul 20% SDS 150ul 20% SDS

q. 50ul 10% APS 100ul 10% APS

r. 25ul TEMED 50ul TEMED

Add APS and TEMED last to stacking solution.

Add stacking solution using 10ml pipette to the top of the bottom plate.

Add comb (avoid bubbles).

Turn on heat block to 100 degrees Celsius.

Prepare Running Buffer (3 Liters)

a. 43.2g Glycine

b. 9g Tris

c. 15mls 20% SDS

H2O to 3 liters

Once gel has polymerized, remove comb gently and evenly.

Boil samples for 5 minutes.

When samples are cool to touch, centrifuge 1minute at 1,000rpm (Don't put samples back on ice).

Assemble buffer chamber:

s. Snap gels onto central core.

t. Put 350mls running buffer into top of core.

u. Let core sit on dry paper towels for 10 minutes to check for leaks.

Load core into buffer chamber.

Put the rest of the buffer into the chamber and core (buffer should be 1ml above bottom of gel).

Load gel (50ul into each lane) and record lanes.

Connect to power supply and turn on. Run on constant volts at 200 Volts until samples reach resolving gel, then turn down to 40 volts and run gel overnight.