Knight:RNA electrophoresis/Denaturing

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.

Reagents

 * RNAse free water (make lots for rinsing glassware and electrophoresis chambers)
 * Add DEPC to final concentration of 0.1%.
 * Incubate 1hr at 37&deg;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&deg;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


 * SYBR Gold

Equipment

 * 37 &deg;C incubator
 * 55 &deg;C water bath
 * Ice water bath
 * Electrophoresis apparatus

Prepare RNase free water

 * 1) Add DEPC to final concentration of 0.1% to H2O
 * 2) Incubate 1hr at 37&deg;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
 * 2) *3 g of PIPES (free acid)
 * 3) *6 g of Bis-Tris (free base)
 * 4) *2 ml of 0.5 M EDTA
 * 5) Treat the solution with final concentration of 0.1% DEPC for 1 hour at 37°C
 * 6) 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&deg;C.

Prepare RNA gel loading buffer

 * 1) Mix the following
 * 2) *95% deionized formamide
 * 3) **Purchase a distilled deionized preparation of formamide and store in small aliquots under nitrogen at -20°C.
 * 4) *0.025% (w/v) bromophenol blue
 * 5) *0.025% (w/v) xylene cyanol FF
 * 6) *5 mM EDTA (pH 8.0)
 * 7) *0.025% (w/v) SDS

Denature RNA samples

 * 1) Mix 10 &mu;L glyoxal reaction mixture with 1-2 &mu;L RNA (up to 10 &mu;g).
 * 2) Also mix 10 &mu;L glyoxal reaction mixture with RNA size marker.
 * 3) Incubate RNA samples at 55&deg;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.

Run gel

 * 1) Add 1-2&mu;L RNA gel loading buffer to glyoxylated RNA samples
 * 2) Immediately load samples. Leave two outside lanes on each side empty.
 * 3) Load RNA size markers on outside lanes of gel.
 * 4) Electrophorese at 5V/cm.

Stain gel

 * 1) Prepare fresh 1:10,000 dilution in RNase free water of SYBR gold.
 * 2) Ensure pH is 7.0-8.5.
 * 3) Pour into staining tray.
 * 4) Place gel in plastic staining container.
 * 5) Shield from light.
 * 6) Agitate gently for 10-40 mins at room temperature.
 * 7) Image with Knight:Gel imager. (Place a clear ruler next to gel to more accurately assess length.)