Knight:RNA electrophoresis/Denaturing: Difference between revisions
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<font color=red>in progress! has not been tested</font> | |||
==Overview== | ==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. | 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. Since this is a denaturing protocol, the RNA can be assessed both for quality and size. | ||
==Materials== | ==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.) | *[[10X BPTE electrophoresis buffer]] (The final pH of this 10x buffer is ~6.5.) | ||
**100 mM PIPES | **100 mM PIPES | ||
Line 16: | Line 23: | ||
**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 | ||
**95% deionized formamide | |||
***Purchase a distilled deionized preparation of formamide and store in small aliquots under nitrogen at -20°C. | |||
**0.025% (w/v) bromophenol blue | |||
**0.025% (w/v) xylene cyanol FF | |||
**5 mM EDTA (pH 8.0) | |||
**0.025% (w/v) SDS | |||
*[[SYBR Gold]] | *[[SYBR Gold]] | ||
===Equipment=== | |||
*37 °C incubator | |||
*55 °C water bath | |||
*Ice water bath | |||
*Electrophoresis apparatus | |||
==Procedure== | ==Procedure== | ||
=== | ===Prepare RNase free water=== | ||
#Prepare by adding the following to 90 ml of distilled H<sub>2</sub>O | #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 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) | ||
Line 27: | Line 53: | ||
#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=== | |||
#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. | |||
===Prepare RNA gel loading buffer=== | |||
#Mix the following | |||
#*95% deionized formamide | |||
#**Purchase a distilled deionized preparation of formamide and store in small aliquots under nitrogen at -20°C. | |||
#*0.025% (w/v) bromophenol blue | |||
#*0.025% (w/v) xylene cyanol FF | |||
#*5 mM EDTA (pH 8.0) | |||
#*0.025% (w/v) SDS | |||
===Denature RNA samples=== | |||
#Mix 10 μL glyoxal reaction mixture with 1-2 μL RNA (up to 10 μg). | |||
#Also mix 10 μL glyoxal reaction mixture with RNA size marker. | |||
#Incubate RNA samples at 55°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. | |||
===Run gel=== | |||
#Add 1-2μL RNA gel loading buffer to glyoxylated RNA samples | |||
#Immediately load samples. Leave two outside lanes on each side empty. | |||
#Load RNA size markers on outside lanes of gel. | |||
#Electrophorese at 5V/cm. | |||
===Stain gel=== | |||
#Prepare fresh 1:10,000 dilution in RNase free water of SYBR gold. | |||
#Ensure pH is 7.0-8.5. | |||
#Pour into staining tray. | |||
#Place gel in plastic staining container. | |||
#Shield from light. | |||
#Agitate gently for 10-40 mins at room temperature. | |||
#Image with [[Knight:Gel imager]]. (Place a clear ruler next to gel to more accurately assess length.) | |||
==Notes== | ==Notes== | ||
*[[Avoiding RNase contamination|Avoid RNase contamination]] | |||
==References== | ==References== | ||
Line 34: | Line 110: | ||
#MolecularCloning [[doi:10.1101/pdb.prot4057|Molecular Cloning: Separation of RNA According to Size: Electrophoresis of Glyoxylated RNA through Agarose Gels]] | #MolecularCloning [[doi:10.1101/pdb.prot4057|Molecular Cloning: Separation of RNA According to Size: Electrophoresis of Glyoxylated RNA through Agarose Gels]] | ||
</biblio> | </biblio> | ||
[[Category:Protocol]] | |||
[[Category:RNA]] | |||
[[Category:In vitro]] |
Latest revision as of 09:50, 6 December 2006
in progress! has not been tested
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. Since this is a denaturing protocol, the RNA can be assessed both for quality and size.
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
- 95% deionized formamide
- Purchase a distilled deionized preparation of formamide and store in small aliquots under nitrogen at -20°C.
- 0.025% (w/v) bromophenol blue
- 0.025% (w/v) xylene cyanol FF
- 5 mM EDTA (pH 8.0)
- 0.025% (w/v) SDS
- 95% deionized formamide
Equipment
- 37 °C incubator
- 55 °C water bath
- Ice water bath
- 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 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
- Treat the solution with final concentration of 0.1% DEPC for 1 hour at 37°C
- Autoclave.
Dilute 10X buffer 10-fold with RNase free H2O.
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.
Prepare RNA gel loading buffer
- Mix the following
- 95% deionized formamide
- Purchase a distilled deionized preparation of formamide and store in small aliquots under nitrogen at -20°C.
- 0.025% (w/v) bromophenol blue
- 0.025% (w/v) xylene cyanol FF
- 5 mM EDTA (pH 8.0)
- 0.025% (w/v) SDS
- 95% deionized formamide
Denature RNA samples
- Mix 10 μL glyoxal reaction mixture with 1-2 μL RNA (up to 10 μg).
- Also mix 10 μL glyoxal reaction mixture with RNA size marker.
- Incubate RNA samples at 55°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.
Run gel
- Add 1-2μL RNA gel loading buffer to glyoxylated RNA samples
- Immediately load samples. Leave two outside lanes on each side empty.
- Load RNA size markers on outside lanes of gel.
- Electrophorese at 5V/cm.
Stain gel
- Prepare fresh 1:10,000 dilution in RNase free water of SYBR gold.
- Ensure pH is 7.0-8.5.
- Pour into staining tray.
- Place gel in plastic staining container.
- Shield from light.
- Agitate gently for 10-40 mins at room temperature.
- Image with Knight:Gel imager. (Place a clear ruler next to gel to more accurately assess length.)