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 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

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 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

Generally denaturing polyacrylamide gels are run at a constant electrical power (Watts). This maintains a certain heated gel temperature during the run. For the 16.5 cm width gels, use 25 W.

Barrick Lab > ProtocolList > ProceduresPolyacrylamideGelElectrophoresis

Contributors to this topic

JeffreyBarrick, AlvaroRodriguez

Topic revision: r2 - 2012-02-18 - 22:47:28 - Main.JeffreyBarrick