Difference: ProceduresPolyacrylamideGelElectrophoresis (4 vs. 5)

Revision 52012-03-12 - JeffreyBarrick

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META TOPICPARENT name="ProtocolList"

Polyacrylamide Gel Electrophoresis

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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.

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
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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.

For nondenaturing gels, use

  Be sure to wash urea out of the wells using a syringe before loading the gel.
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  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.

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
 
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