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.

Spacer Width Gel Height Gel Width Gel Vol TEMED Vol 10% APS Vol
1 mm 28 cm 16.5 cm 50 ml 20 l 400 l
1 mm 14.5 cm 16.5 cm 25 ml 10 l 200 l

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 4C. 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.

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 l
1 mm 16.5 cm 15 mm 4 mm 20 60 l
1 mm 16.5 cm 15 mm 2 mm 30 30 l

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

36 ml Deionized formamide
4 ml 10x TBE Buffer
16 mg Bromophenol Blue

Makes 40 ml.

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

10x TBE Buffer

108 g Tris base
55 g Boric acid
40 ml 0.5 M EDTA, pH 8.0

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.
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Topic revision: r4 - 12 Mar 2012 - 15:40:16 - Main.JeffreyBarrick
 
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