Hazards
Acrylamide monomer is a potent cumulative neurotoxin.
Protection
Do not mouth pipette acrylamide solutions, use care and wear gloves while handling unpolymerized solutions that contain it. Use in a well ventilated area, and report any spills. Stock solutions should be kept in a fume hood.
Waste
Gel remains and used gel should be disposed as hazardous waste.
Spill Clean-up
* Acrylamide (30 or 40% with Acryl
to Bis-Acryl ratio 37.5:1)
* TEMED
* 10% Ammonium Persulfate
* 1.5 M Tris-Cl pH 8.8
* 0.5 M Tris-Cl pH 6.8
* 10% SDS
* Tris, Glycine, SDS
* Glycerol, 2-ME, 1% bromophenol blue
* Mini-Protean electrophoresis cell
* Power pack capable of at least 200 V, 350 mA
5 x Running Buffer, pH should be
around 8.3. For 1 L, in di H2O:
Tris base: 9g
Glycine: 72g
SDS: 5g
Store at 4 ¡C (may come out of solution
- if so warm to room temp)
2 x Reducing (Loading) buffer:
Recipe for 50 ml stock
12.5 ml of 125 mM Tris pH 6.8 (0.5 M)
12.5 ml Glycerol (25%)
12.5 ml 2.5% SDS (10%)
5 ml 10% 2-ME (Note: massive excess)
7.5 ml di H2O
Resolving gel:
For 8 ml (volumes for 10 ml are in mini-gel instruction book)
To make a 12% resolving gel:
3.5 ml di H2O
2 ml 1.5 M Tris-Cl pH 8.8
80 µl 10% SDS
2.4 ml Acryl/Bis (40% stock)
Store 4¡C - let aliquot warm to room
temp.
40 µl 10% ammonium persulfate
Store aliquots at -20¡C.
4 µl TEMED add last and store at
4¼C
Total volume ~ 8 ml
To make a 7.5% resolving gel:
4.4 ml di H2O
2 ml 1.5 M Tris-Cl pH 8.8
180 µl 10% SDS
1.5 ml Acryl/Bis (40% stock)
Store 4¡C - let aliquot warm to room
temp.
140 µl 10% ammonium persulfate
Store aliquots at -20¡C
4 µl TEMED add last and store at
4¼C
Total volume ~ 8 ml
Procedure: Assemble gel plates as
described in instruction book. Add reagents, TEMED last, and pour, using a
pasteur or a 5 ml pipet. This is the resolving gel, so do not fill the plates -
only fill to within about 0.5 - 1 cm from the top of the short plate. Layer
about 0.5 ml of isopropanol onto the gel, this will help form a straight edge
for the upper gel surface, and speed up polymerization.
Allow to set for about 30-45 mins.
Stacking gel
For 5 ml of 4% gel
3.5 ml di H2O
1.25 ml 0.5 M Tris-Cl pH 6.8
50 µl 10% SDS
0.5 ml Acryl/Bis (40% stock)
Store 4¡C - let aliquot warm to room
temp.
25 µl 10% ammonium persulfate
Aliquots at -20¡C
5 µl TEMED
Store at 4¡C, add last
Total Volume ~ 5ml
Procedure: pour off isopropanol,
and wick beads of liquid away using kimwipes. Wash surface of gel with di water
and wick that away also. Pour stack gel and assemble comb into place. This gel
takes about 30-45 mins to set.
Remove comb (gently) and wash the wells with running buffer. Assemble holder
into running tank.
Do not pre-run these gels (the
different concentrations and pH of the stack and resolving gel will be lost if
the gel is pre-run - this gradient is important for the stacking effect).
Prepare the gel close to a time when the samples will be ready (within a few
hours).
Load the gel using a fine pipetman tip (or a hamilton syringe). well loading
volumes are variable, depending on spacer size, but the 0.75 space, 10 tooth
comb will hold about 20 - 25 µl maximum.
Connect the gel box to a power pack. Typically we run these gels fast - at
about 200 volts for 45min - 1 hr. Stacking allows you to do this without
loosing resolution. However, you can probably improve resolution by slowing
this down somewhat (e.g. 150 V for 1-2 hr).
We have an occasional problem with leaking, A) from the plates while the gel is poured and B) from the inner chamber of the running tank (the inner cooling core) during electrophoresis. For the former problem, ensure that the gel plates are assembled correctly, and use a layer (1 or more sheets) of parafilm on the casting stand beneath the gel plates. For the latter problem, be careful when assembling the gel into the runing chamber that it fits correctly against the rubber gaskets of the inner cooling core.