Difference: ProceduresPolyacrylamideGelElectrophoresis (5 vs. 6)

Revision 62012-03-13 - JeffreyBarrick

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META TOPICPARENT name="ProtocolList"

Polyacrylamide Gel Electrophoresis

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

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For denaturing gels, use the 2× formamide sample loading buffer.
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For denaturing gels, use the 2x formamide sample loading buffer.
 
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For nondenaturing gels, use
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For nondenaturing gels, use the 5x glycerol sample loading buffer.
 
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Be sure to wash urea out of the wells using a syringe before loading the gel.
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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
 
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