Knight:RNA electrophoresis/Denaturing
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(Difference between revisions)
| Line 4: | Line 4: | ||
==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 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
- 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.
