Polyacrylamide gels are chemically cross-linked gels formed by
the polymerization of acrylamide with a cross-linking agent, N,N'-methylenebisacrylamide
(Bis). The reaction is a free radical polymerization, carried
out with ammonium persulfate as the initiator and N,N,N',N'-tetramethylenediamine
(TEMED) as the catalyst.
MATERIALS AND SOLUTIONS
30% Acrylamide:Bis Solution (29:1)
Acrylamide ------------------------------------------ 29 g
Bis-acrylamide --------------------------------------- 1 g
Add distilled H2O to make a final volume of -------- 100 ml
Dissolve the acrylamide and Bis-acrylamide in distilled water
for 1 hour.
Filter the solution through a 0.4 um filter to remove undissolved
particles.
Store in a brown bottle at 4oC for 1 month.
Caution: Acrylamide is a neurotoxin!
10% Ammonium persulfate (10 ml)
Ammonium persulfate -------------------------------- 1 g
Add distilled H2O to make a final volume of -------- 10 ml
5 x TBE Buffer (1 liter)
0.32 M Boric acid ----------------------------------- 10 g
0.5 M Trizma base ---------------------------------- 30.25 g
10 mM EDTA-Na2 --------------------------------- 1.86 g
Add distilled H2O to make a final volume of ------- 1 liter
Adjust pH to 8.6.
50 X TAE Buffer (100 ml)
0.4 M Trizma base ------------------------------- 24.22 g
10 mM EDTA-Na2 ------------------------------ 1.86 g
Glacial acetic acid -------------------------------- 13 ml
Add distilled H2O to make a final volume of ----- 100 ml
Adjust pH to 8.0.
Ethidium Bromide Solution (10 mg/ml)
Ethidium bromide --------------------------------- 100 mg
Add distilled H2O to make a final volume of ----- 10 ml
Store in a brown bottle at 4oC.
10 X Glycerol Dye Mix (10 ml)
Glycerol ------------------------------------------ 6 ml
0.2% Bromophenol blue -------------------------- 0.4 ml of 5% Bromophenol blue
0.2% Xylene cyanol FF --------------------------- 0.4 ml of 5% Xylene cyanol FF
5 X TBE (or 5 X TAE) ------------------------ 2 ml
Distilled H2O ------------------------------------- 1.2 ml
10 X Ficoll Dye Mix (5 ml)
50% Ficoll type 400L (MW 400,000) ----------- 2.5 g
0.14% Bromophenol blue ------------------------ 0.14 ml of 5% Bromophenol blue
0.14% Xylene cyanol FF ------------------------- 0.14 ml of 5% Xylene cyanol FF
5 X TBE (or 5 X TAE) ------------------------ 2 ml
Add distilled H2O to make a final volume of ----- 5 ml
PROCEDURES
1. Prepare the gel apparatus.
2. Mix the polyacrylamide gel solution in a beaker.
3. Add 85 ul of TEMED (N,N,N',N'-tetramethylenediamine).
4. Immediately, pour the gel mixture slowly into the previously prepared gel
form, until the liquid level reaches the top of the upper glass
plate.
5. Insert the comb between the two glass plates and clamp in place
with a small spring clip.
6. Dribble a little extra gel mixture over the junction between
the comb and the glass plates, using a pipette, to compensate
for volume shrinking upon gelation and small leaks. Repeat as
necessary.
7. Let the gel stand 1-2 hours or overnight to solidify.
8. Remove the comb by gently wriggling it and lifting out.
Wash unpolymerized acrylamide out of the wells by squirting with
distilled water.
Remove the water from the wells.
Remove the spring clips and bottom space from the gel form.
9. Attach the ears to the gel form.
Place narrow lines of high vacuum grease from each edge of the
glass plate.
10. Clamp the gel form to the gel cabinet with two large spring
clamps.
11. Fill the lower and upper tank with 1 X TBE or 1 X TAE.
The
running buffer must be the same as the buffer in which the gel
was cast.
12. Use a bent syringe needle to perforate and remove air bubbles
trapped in the space where the bottom spacer was originally located.
Remove any air bubbles from the wells of the gel by squirting
with running buffer.
13. Allow the gel to equilibrate with the buffer in the gel cabinet
for 1-24 hours.
14. Pre-electrophorese polyacrylamide gels for 2 hours, at a voltage
of 3 volts/cm.
During pre-electrophoresis, the current will decrease and then
become essentially constant.
15. After the pre-electrophoresis is finished, turn off the high
voltage.
Load the DNA samples (mixed with Glycerol Dye Mix or Ficoll Dye
Mix) in the wells.
To get sharp bands, the DNA sample should be 1 mm or less in height
after loading.
16. Set the high voltage to the desired value and start the electrophoresis
run.
17. After the electrophoresis is finished, turn off the power
supply.
Pour off the buffer in the buffer tanks.
18. Remove the side binder clamps and pry the gel from the gel
cabinet by placing a metal spatula between the back glass plate
and the gel cabinet and twisting gently.
Separate the top glass plate from the gel.
19. Place the glass plate holding the polyacrylamide gel in 100
ml of a 1 X TBE (or, 1 X TAE) solution and 15 ul of Ethidium Bromide
Solution (10 mg/ml).
22. Destain the gel 15-30 minutes in distilled water.
23. Illuminate analytical gels with short wavelength UV light
(254 nm) generated by Mineral-light model R-52.
24. Illuminate preparative gels with long wavelength UV light
(365 nm) generated by Mineral-light model R-56, to prevent nicking
of the DNA/dye complexes.
25. Photograph the gel
NOTES
Polyacrylamide gels must be pre-electrophoresed before use, in order to eliminate charged impurities (assumed to be ammonium
persulfate derivatives) from the gel matrix. If this is not done,
the charged impurities will form complexes with the DNA fragments,
changing their electrophoretic mobilities in a non-reproducible
manner.
The polymerization reaction requires 24 hours to reach completion.
For accurate, quantitative determination of DNA electrophoretic
mobilities, polyacrylamide gel should be aged 24 hours.
When Joule heating becomes important, the temperature near the
center of the gel is higher than the temperature near the edges.
The resulting difference in the viscosity of the solvent causes
the DNA bands to migrate faster near the center of the gel, giving
a smiling effect when the same sample is loaded in all the wells.