- 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].