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.

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

Formamide sample loading buffer.

Be sure to wash urea out of the wells using a syringe before loading the gel.

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 Loading Buffer

Makes 40 ml.

The final concentration of EDTA in this buffer at 1x is 10 mM.

10x TBE Buffer

Add ddH₂O to 1 L.

0.5 M EDTA, pH 8.0

1. Dissolve 186.1 g Na₂EDTA in 700 ml ddH₂O.
2. Adjust pH to 8.0 with 10 M NaOH.
3. Add ddH₂O to 1 L.