Polyacrylamide Gel Electrophoresis
Our gel rigs and supplies are from
CBS Scientific.
The
National Diagnostics Website has very helpful background on RNA/DNA polyacrylamide gels.
Pouring the Gel
For
denaturing urea gels, we use the
SequaGel system. Check out the link to determine how to mix up a gel of the proper percentage.
For
nondenaturing gels, use the
AccuGel system. The concentrated stock solution is 40%. Dilute the stock to get the desired gel percentage in your final volume. Add 1/10th volume 10x TBE buffer, then ddH
2O for the rest of the volume.
Spacer Width |
Gel Height |
Gel Width |
Gel Vol |
TEMED Vol |
10% APS Vol |
1 mm |
28 cm |
16.5 cm |
50 ml |
20 µl |
400 µl |
1 mm |
14.5 cm |
16.5 cm |
25 ml |
10 µl |
200 µl |
TEMED is N,N,N',N'-tetramethylethylenediamine. APS is ammonium persulfate. Together, they create the radicals to initiate polymerization of the gel. TEMED and 10% APS should be stored at 4°C. APS solutions should be freshly prepared for best results.
Acrylamide is a neurotoxin before it is polymerized. You are working with it in a liquid solution where spills may happen. Wear gloves, a lab coat, and safety glasses when working with polyacrylamide gels.
Loading the Gel
For denaturing gels, use the 2× formamide sample loading buffer.
For nondenaturing gels, use
Be sure to wash urea out of the wells using a syringe before loading the gel.
Spacer Width |
Gel Width |
Well Height |
Well Width |
# Wells in Comb |
Max Sample Vol |
1 mm |
16.5 cm |
15 mm |
8 mm |
12 |
120 µl |
1 mm |
16.5 cm |
15 mm |
4 mm |
20 |
60 µl |
1 mm |
16.5 cm |
15 mm |
2 mm |
30 |
30 µl |
For sharp bands, you should load to much less than the maximum well volume.
Running the Gel
You will need a high voltage power supply to run the large vertical polyacrylamide gels. Generally denaturing gels are run at a constant electrical power (Watts). This maintains a certain heated gel temperature during the run. For the 16.5 cm x 28.5 cm gels, use 25 W. Using a higher voltage can cause excessive heating that will crack the glass plates.
Reagents
All reagents should be prepared with RNAse, DNase free water.
2x Formamide Loading Buffer
32 ml |
Deionized formamide |
8 ml |
10x TBE Buffer |
16 mg |
Bromophenol Blue |
Makes 40 ml. Use for
denaturing gels.
Note: Many recipes for this do not add the TBE buffer. This is fine if you are loading straight from a reaction which has buffer in it, but may cause problems if your sample is in pure H
2O (for example, after EtOH precipitation).
The final concentration of EDTA in this buffer at 1x is 10 mM.
5x Glycerol Loading Buffer
20 ml |
10x TBE Buffer |
20 ml |
Glycerol |
16 mg |
Bromophenol Blue |
Makes 40 ml. Use for
nondenaturing gels.
Note: Many recipes for this do not add the TBE buffer or substitute sucrose for glycerol.
10x TBE Buffer
108 g |
Tris base |
55 g |
Boric acid |
40 ml |
0.5 M EDTA, pH 8.0 |
Add ddH
2O to 1 L.
0.5 M EDTA, pH 8.0
- Dissolve 186.1 g Na2EDTA in 700 ml ddH2O.
- Adjust pH to 8.0 with 10 M NaOH.
- Add ddH2O to 1 L.