Knight:RNA electrophoresis/Denaturing: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
No edit summary |
||
Line 4: | Line 4: | ||
==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 | ||
Line 22: | Line 23: | ||
**Autoclave for 15 mins at 15 psi. | **Autoclave for 15 mins at 15 psi. | ||
*[[SYBR Gold]] | *[[SYBR Gold]] | ||
===Equipment=== | |||
*Electrophoresis apparatus | |||
==Procedure== | ==Procedure== | ||
=== | ===Prepare RNase free water=== | ||
#Add DEPC to final concentration of 0.1% to H<sub>2</sub>O | |||
#Incubate 1hr at 37°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) | ||
Line 31: | Line 40: | ||
#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°C. | |||
==Notes== | ==Notes== |
Revision as of 14:23, 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
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
- Add DEPC to final concentration of 0.1% to H2O
- Incubate 1hr at 37°C.
- Autoclave for 15 mins at 15 psi.
Prepare 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.
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°C.