Knight:RNA electrophoresis/Denaturing

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==Materials==
==Materials==
 +
===Reagents===
*[[10X BPTE electrophoresis buffer]] (The final pH of this 10x buffer is ~6.5.)
*[[10X BPTE electrophoresis buffer]] (The final pH of this 10x buffer is ~6.5.)
**100 mM PIPES
**100 mM PIPES
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**Autoclave for 15 mins at 15 psi.
**Autoclave for 15 mins at 15 psi.
*[[SYBR Gold]]
*[[SYBR Gold]]
 +
 +
===Equipment===
 +
*Electrophoresis apparatus
==Procedure==
==Procedure==
-
===Make up BPTE electrophoresis buffer===
+
===Prepare RNase free water===
 +
#Add DEPC to final concentration of 0.1% to H<sub>2</sub>O
 +
#Incubate 1hr at 37&deg;C.
 +
#Autoclave for 15 mins at 15 psi.
 +
 
 +
===Prepare BPTE electrophoresis buffer===
#Prepare by adding the following to 90 ml of distilled H<sub>2</sub>O
#Prepare by adding the following to 90 ml of distilled H<sub>2</sub>O
#*3 g of PIPES (free acid)
#*3 g of PIPES (free acid)
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#Treat the solution with final concentration of 0.1% DEPC for 1 hour at 37°C
#Treat the solution with final concentration of 0.1% DEPC for 1 hour at 37°C
#Autoclave.
#Autoclave.
 +
 +
===Prepare glyoxal reaction mixture===
 +
#6mL DMSO
 +
#2mL deionized glyoxal
 +
#1.2mL of 10X BPTE electrophoresis buffer
 +
#0.6mL of 80% glycerol
 +
 +
Divide into small aliquots and store at -70&deg;C.
==Notes==
==Notes==

Revision as of 16:23, 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

Reagents

  • 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
  • RNAse free water (make lots for rinsing glassware and electrophoresis chambers)
    • Add DEPC to final concentration of 0.1%.
    • Incubate 1hr at 37°C.
    • Autoclave for 15 mins at 15 psi.
  • SYBR Gold

Equipment

  • Electrophoresis apparatus

Procedure

Prepare RNase free water

  1. Add DEPC to final concentration of 0.1% to H2O
  2. Incubate 1hr at 37°C.
  3. Autoclave for 15 mins at 15 psi.

Prepare 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.

Prepare glyoxal reaction mixture

  1. 6mL DMSO
  2. 2mL deionized glyoxal
  3. 1.2mL of 10X BPTE electrophoresis buffer
  4. 0.6mL of 80% glycerol

Divide into small aliquots and store at -70°C.

Notes

References

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