RNA Gel Electrophoresis
- Mix up the gel.
10X E Reagent Amount Na2HPO4 25.56g NaH2PO4·H2 2.76g H2O to 1 L
For a 150 ml 1.2% agarose gel, add 15 ml of 10X E to 110.7 ml of H2O. Add 1.8 g of agarose and dissolve by heating. Allow the gel to cool in the hood until it reaches 65° and then add 24.3 ml of 37% formaldehyde. Mix and immediately pour the gel.
- Before preparing the RNA samples, wash the buffer recirculation pump and hoses by circulating with 0.2N NaOH for 30 minutes and then rinsing with ddH2O.
- Prepare the RNA samples
Solution A Reagent Amount deionized formamide 500 µl formaldehyde 162 µl 10X E 100 µl bromophenol blue “a pinch”
The RNA should be 2 to 15 µg in 4 µl or less. To each RNA sample add 3 volumes of Solution A.
- Heat the samples at 60° for 5 minutes to denature the RNA.
- Quick cool the samples on ice.
- Load the RNA for electrophoresis. Load one lane of RNA standards. We use GibcoBRL #15620-016 .24 to 9.5 kb RNA Ladder, and add 3 µg per lane. Be sure to leave an empty lane between the RNA Ladder and the other lanes.
Low Ionic Strength Buffer [Stock] Reagent Amount [Final] 10X E 100 ml 1X 37% formaldehyde 81 ml 3% — H2O 819 ml —
Since the gel and buffer contain formaldehyde, the gel must be run in the hood. The buffer must be recirculated when using low ionic strength buffer.
- Run the gel overnight at 40V.
- Only the lane containing the RNA ladder should be stained and photographed. Cut the appropriate lane away from the rest of the gel. Drive off the formaldehyde by pouring 500 ml of 60° H2O over the gel and shaking at room temperature for 5 minutes. Stain the lane in 0.1M NH4OAc, 1 µg/ml EtBr for 30 minutes. Destain in H2O for 30 minutes.
- Transfer to Ambion BrightStar-Plus and crosslink by following the protocol in the Ambion Technical Bulletin #169 . We have had good results with baking the membrane.
- Hybridize the filter following the protocol for using Ambion’s ULTRAhyb [old link no longer works].
This page is, as always, Copyright (c) 1999 by Craig D. Amundsen. All rights reserved.