Determination of RNA fragment lenghts in Northern Blots without labelled markers

Determination of RNA fragment lenghts in Northern Blots without labelled markers
From Fermentas

1.) Load the RNA marker on the Northern Gel beside your samples

Important: Use the same loading dye for the marker and your samples AND load same volumes. Adjust the volumes with DEPC water

2.) Run the gel and blot the RNA. Crosslink the RNA on the membrane
3.) Cut off the marker lane

Possibility A: Staining with methylene blue

4.) Put the membrane containing the marker lane into a clean, flat bowl. Add methylene blue solution. The membrane must be completely covered (ideally, the gauge should be around 0.8 cm)

5.) Shake the bowl very carefully for about 5-10 min

6.) When the RNA marker bands appear blue, discard the methylene blue solution and wash the membrane with tap water (optionally, 2 x 30 s). The methylene blue solution can be reused several times (store at 4°C)
7.) The marker lane can be a) scanned beside the developed blot or b) mark with a pen the marker bands on the blot (pen writings are mostly visible on fluorescence scanners)
Methylene blue solution: e.g. the Methylene blue solution from MRC (Molecular Research Center), distributed by Fermentas

Possibility B: Visualizing with UV light

4.) Put the membrane containing the marker lane on an UV screen. Mark with a pen the marker bands and the marker lane can be scanned beside the developed blot