Solutions

1. 30% acrylamide/.8%bisacrylamide 30g Acrylamide

(store in brown light sensitive container) .8g Bisacrylamide

Bring up to 100ml.

2. Lower Tris:HCl SDS (ph8.8) 4x TO 300 ml. _dH₂O add:

91g Tris Base(1.5MTris:Cl,final)

2g SDS (.4% SDS final)

adjust pHto 8.8 with 1N Hcl

Add _dH₂O to 500ml.

Filter through a .45m l filter and store at 4° C

3. Upper Tris:HCl SDS (pH6.8) 4x to 40 ml _dH₂O add:

6.05g Tris Base(.5M Tris:Cl, final)

0.4g SDS ( .4% final) Adjust pH to 6.8 with 1N Hcl

Add _dH₂O to 100 ml.

Filter through a .45m l filter and store at 4° C

4.Amonium Persulfate: 10%solution(made semi-fresh)

5 Temed( light sensitive) GIBCO

Store all above solutions on the cold room or 4° c

Total volume: 10 ml

10X RUNNING BUFFER: 30.3 g Tris-base

144.0 g glycine

10.0 g SDS

2X SAMPLE BUFFER: 1.25 ml Tris-HCl pH 6.8

4.0 ml 10% SDS

2.0 ml glycerol

make up to 10 ml with H2O.

FIXING SOLUTION: 50% methanol

40% H2O

10% acetic acid (ethanoic acid)

STAINING SOLUTION: same as above except 0.5 mg/ml Coomassie blue

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Lubriseal layer

max 1mm thick

1. Assemble glass plate sandwich using two clean glass plates and two .75-mm spacers.

(be sure and align the plates and the spacers well.)

2. (Optional) Use lubriseal and coat the bottom edge of the plate-spacer apparatus. This helps prevent the acrylamide from leaking if the caster is not set up properly.

3. Place the plates in the caster and tighten opposing screws carefully. (glass plates are very easily broken.)

4. Then when screws are tight depress the glass plates using the knobs on the side of the dual gel caster. (It may not be necessary to turn the knobs the entire way down. You can controll the amount of pressure applied by the degree to which you turn the knobs.) (Also be sure that you have used to small metal clamps for the top of the glass plates, Otherwise; it will leak, ruining the gel)

5. Make up solution of Ammonium persulfate 10%.

6. First pour the resolving Gel (bottom layer) Be sure to use Lower Tris when making the resolving part. Wait 1 min then layer the top of the gel with saturated butyl alcohol. (this will help the gel to solidify quickly, acrylimide polymerizes quicker in the absence of air)

7. Wait another 15-20 min for the gel to polymerize. Pour off the top layer of butyl alcohol and rinse with distilled water. Dry off as much of the water as possible.

8. Next pour the pour the resolving gel (Using Upper Tris). Pour till the space is full then put in the appropriate comb. ( The difference in the pH of the two layers is what causes the stacking of the protein. Thus, if the protein is not stacking properly check the pH of the buffers.)

9. Allow the top portion to solidify and then carefully remove the comb.