Difference: ProceduresPolyacrylamideGelElectrophoresis (4 vs. 5)

Revision 52012-03-12 - JeffreyBarrick

META TOPICPARENT	name="ProtocolList"

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

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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.

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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.

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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	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

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Formamide sample loading buffer.

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For denaturing gels, use the 2× formamide sample loading buffer.

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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.

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2x Loading Buffer

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2x Formamide Loading Buffer

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36 ml	Deionized formamide
4 ml	10x TBE Buffer

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32 ml	Deionized formamide
8 ml	10x TBE Buffer

16 mg

Bromophenol Blue

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Makes 40 ml.

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Makes 40 ml. Use for denaturing gels.

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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.

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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 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.

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