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 µl	200 µl
1 mm	28 cm	16.5 cm	50 ml	50 µl	400 µl
1.5 mm	14.5 cm	16.5 cm	30 ml	30 µl	240 µl
1.5 mm	28 cm	16.5 cm	75 ml	75 µl	600 µl

Add 10 µl of TEMED per 10 ml volume, then 50 µl 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 4°C. 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 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 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 µl	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

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

Barrick Lab > ProtocolList > ProceduresPolyacrylamideGelElectrophoresis

Contributors to this topic

JeffreyBarrick, AlvaroRodriguez

Topic revision: r8 - 2013-06-07 - 16:27:32 - Main.AlvaroRodriguez