Knight:RNA electrophoresis/Denaturing

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==Materials==
==Materials==
===Reagents===
===Reagents===
 +
*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.
*[[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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**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 size marker
*RNA gel loading buffer
*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]]
*[[SYBR Gold]]
===Equipment===
===Equipment===
 +
*37 °C incubator
 +
*55 °C water bath
 +
*Ice water bath
*Electrophoresis apparatus
*Electrophoresis apparatus
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===Prepare BPTE electrophoresis buffer===
===Prepare BPTE electrophoresis buffer===
-
#Prepare by adding the following to 90 ml of distilled H<sub>2</sub>O
+
#Prepare 10X buffer 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)
#*6 g of Bis-Tris (free base)
#*6 g of Bis-Tris (free base)
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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.
 +
 +
Dilute 10X buffer 10-fold with RNase free H<sub>2</sub>O.
===Prepare glyoxal reaction mixture===
===Prepare glyoxal reaction mixture===
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Divide into small aliquots and store at -70&deg;C.
Divide into small aliquots and store at -70&deg;C.
 +
 +
===Denature RNA samples===
 +
#Mix 10 &mu;L glyoxal reaction mixture with 1-2 &mu;L RNA (up to 10 &mu;g).
 +
#Also mix 10 &mu;L glyoxal reaction mixture with RNA size marker.
 +
#Incubate RNA samples at 55&deg;C for 1 hr.
 +
#Chill RNA samples for 10 mins in ice water.
 +
#Centrifuge 5 seconds to collect liquid at the bottom of the tubes.
 +
 +
===Cast gel===
 +
Do this step during 1 hr denaturation of samples.
 +
 +
#Cast 1.5% agarose gel in 1X BPTE electrophoresis buffer.
 +
#Use a comb with at least 4 extra lanes for size markers and running dyes.
 +
#Place gel in chamber.
 +
#Cover with 1X BPTE electrophoresis buffer to cover gel to a depth of 1mm.
==Notes==
==Notes==

Revision as of 17:31, 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

  • 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.
  • 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 size marker
  • RNA gel loading buffer

Equipment

  • 37 °C incubator
  • 55 °C water bath
  • Ice water bath
  • 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 10X buffer 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.

Dilute 10X buffer 10-fold with RNase free H2O.

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.

Denature RNA samples

  1. Mix 10 μL glyoxal reaction mixture with 1-2 μL RNA (up to 10 μg).
  2. Also mix 10 μL glyoxal reaction mixture with RNA size marker.
  3. Incubate RNA samples at 55°C for 1 hr.
  4. Chill RNA samples for 10 mins in ice water.
  5. Centrifuge 5 seconds to collect liquid at the bottom of the tubes.

Cast gel

Do this step during 1 hr denaturation of samples.

  1. Cast 1.5% agarose gel in 1X BPTE electrophoresis buffer.
  2. Use a comb with at least 4 extra lanes for size markers and running dyes.
  3. Place gel in chamber.
  4. Cover with 1X BPTE electrophoresis buffer to cover gel to a depth of 1mm.

Notes

References

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