Knight:RNA electrophoresis/Native

Overview
Electrophoresis permits assessment of RNA by size and amount. However, in native gels RNA electrophoretic mobility will depend on not only the size of the RNA but also its secondary structure. Since this is a native gel you are more likely to see a smear of RNA and/or multiple bands because of RNA secondary structure.

Procedure
The procedure for running a native RNA gel is very similar to that for running a DNA agarose gel. Here are a few things to note.


 * 1) Generally load 1 &mu;g and 2.5 &mu;g samples.
 * 2) Use TBE buffer (89 mM Tris-HCl pH 7.8, 89 mM borate, 2 mM EDTA)?
 * 3) *TAE buffer also seems to work equally well in my hands for ~1000bp transcripts.
 * 4) An aliquot of intact RNA should always be run as a positive control to rule out unusual results due to gel artifacts.
 * 5) Run the gel at low voltage (reports of 5-6 V/cm or 8V/cm are common as measured between the electrodes).
 * 6) *I tried ~65V total.
 * 7) Use an RNase free aliquot of loading buffer (i.e. made with RNase free water).
 * 8) RNA Ladder
 * 9) *2 &mu;L RNA ladder
 * 10) *2 &mu;L loading dye
 * 11) *8 &mu;L H2O
 * 12) Also run a DNA ladder

Staining

 * 1) Dilute SYBR Gold 10,000-fold into 1X running buffer.
 * 2) *Note that SYBR gold is preferable to SYBR green II
 * 3) Incubate agarose gel in staining solution for 40 minutes.
 * 4) Visualize on gel imager. (No destaining needed).