Knight:RNA electrophoresis/Denaturing

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==Materials==
==Materials==
-
*[[10X BPTE electrophoresis buffer]]
+
*[[10X BPTE electrophoresis buffer]] (The final pH of this 10x buffer is ~6.5.)
-
*[[SYBR Gold]]
+
**100 mM PIPES
 +
**300 mM Bis-Tris
 +
**10 mM EDTA
*HPLC grade or better DMSO
*HPLC grade or better DMSO
*Glyoxal
*Glyoxal
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**1.2mL of 10X BPTE electrophoresis buffer
**1.2mL of 10X BPTE electrophoresis buffer
**0.6mL of 80% glycerol
**0.6mL of 80% glycerol
 +
*RNA gel loading buffer
 +
*[[SYBR Gold]]
==Procedure==
==Procedure==
 +
===Make up BPTE electrophoresis buffer===
 +
#Prepare by adding the following to 90 ml of distilled H<sub>2</sub>O
 +
#*3 g of PIPES (free acid)
 +
#*6 g of Bis-Tris (free base)
 +
#*2 ml of 0.5 M EDTA
 +
#Treat the solution with final concentration of 0.1% DEPC for 1 hour at 37°C
 +
#Autoclave.
==Notes==
==Notes==
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==References==
==References==
<biblio>
<biblio>
-
#MolecularCloning [[Molecular Cloning]]
+
#MolecularCloning [[doi:10.1101/pdb.prot4057|Molecular Cloning: Separation of RNA According to Size: Electrophoresis of Glyoxylated RNA through Agarose Gels]]
</biblio>
</biblio>

Revision as of 17:00, 5 December 2006

Contents

Overview

Electrophoresis permits assessment of RNA by size and amount. In general, electrophoresis of RNA is done as a step prior to Northern analysis. However, this protocol is for visualizing the RNA in the gel.

Materials

  • 10X BPTE electrophoresis buffer (The final pH of this 10x buffer is ~6.5.)
    • 100 mM PIPES
    • 300 mM Bis-Tris
    • 10 mM EDTA
  • HPLC grade or better DMSO
  • Glyoxal
    • Commercially available stock solutions of glyoxal contain both hydrated forms of glyoxal and oxidation products that can degrade RNA. These must be removed.
  • Glyoxal reaction mixture (divide into small aliquots and store at -70°C)
    • 6mL DMSO
    • 2mL deionized glyoxal
    • 1.2mL of 10X BPTE electrophoresis buffer
    • 0.6mL of 80% glycerol
  • RNA gel loading buffer
  • SYBR Gold

Procedure

Make up BPTE electrophoresis buffer

  1. Prepare by adding the following to 90 ml of distilled H2O
    • 3 g of PIPES (free acid)
    • 6 g of Bis-Tris (free base)
    • 2 ml of 0.5 M EDTA
  2. Treat the solution with final concentration of 0.1% DEPC for 1 hour at 37°C
  3. Autoclave.

Notes

References

  1. Molecular Cloning: Separation of RNA According to Size: Electrophoresis of Glyoxylated RNA through Agarose Gels [MolecularCloning]
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