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 ddH2O for the rest of the volume.

Spacer Width Gel Height Gel Width Gel Vol TEMED Vol 10% APS Vol
1 mm 14.5 cm 16.5 cm 25 ml 25 l 200 l
1 mm 28 cm 16.5 cm 50 ml 50 l 400 l
1.5 mm 14.5 cm 16.5 cm 30 ml 30 l 240 l
1.5 mm 28 cm 16.5 cm 75 ml 75 l 600 l

Add 10 l of TEMED per 10 ml volume, then 50 l of 10% w/v APS per 10 ml volume to initiate polymerization of the gel. Pour it quickly into the glass plates with spacers clamped between them.

TEMED is N,N,N',N'-tetramethylethylenediamine. APS is ammonium persulfate. Together, they create the radicals to polymerize the gel. TEMED and 10% APS should be stored at 4C. APS solutions should be freshly prepared for best results.

For gels that are 1 mm or less in thickness, you can prop the glass plates horizontally and pour the gel without using a casting boot. Surface tension will keep the gel solution from coming out the bottom.

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 2x formamide sample loading buffer.

For nondenaturing gels, use the 5x glycerol sample loading buffer.

Be sure to wash buffer out of the wells using a syringe immediately 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 35 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 H2O (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.

6x Urea Loading Buffer

24 g Urea
2 ml 0.5 M EDTA
100 l 1 M Tris pH 7.5
16 mg Bromophenol Blue

Add ddH2O to 50 ml. Use for Use for denaturing gels.

Note: Bromophenol Blue is optional.

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: r8 - 07 Jun 2013 - 16:27:32 - Main.AlvaroRodriguez