Knight:RNA electrophoresis/Denaturing: Difference between revisions
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==Materials== | ==Materials== | ||
*[[10X BPTE electrophoresis buffer]] | *[[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 | *HPLC grade or better DMSO | ||
*Glyoxal | *Glyoxal | ||
| Line 14: | Line 16: | ||
**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== | ||
| Line 21: | Line 32: | ||
==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 14:00, 5 December 2006
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
- 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
- Treat the solution with final concentration of 0.1% DEPC for 1 hour at 37°C
- Autoclave.
