Journal of Biochemical and Biophysical Methods
Volume 68, Issue 2, 31 August 2006, Pages 139-143
Short noteAn improved method for Southern DNA and Northern RNA blotting using a Mupid®-2 Mini-Gel electrophoresis unit
Abstract
An improved method for Southern DNA and Northern RNA blotting using the Mupid®-2 Mini-Gel System is described. We get sharp and clear bands in Southern and Northern blotting after only 30 min short gel electrophoresis instead of the several hours large gel electrophoresis of conventional methods. The high electrical voltage with a pulse-like current of the Mupid®-2 Mini-Gel System also allows reduction of the amount of formaldehyde, a harmful reagent, from the gel running buffer in RNA blotting. This minor modification of DNA and RNA blotting technique enables us to perform the complete experimental procedure more quickly economically in less space, than conventional Southern and Northern blotting, as well as using an extremely small amount of formaldehyde in RNA blotting.
Section snippets
Southern blotting
Genomic DNA is obtained from human blood samples. Genomic DNA (10 μg) is digested with the restriction enzyme and precipitated with ethanol, and the concentration is quantified by means of a NanoDrop spectrophotometer (NanoDrop Tech). Then 7.5 μg of the DNA is electrophoresed in an agarose gel, using the usual TAE buffer (40 mM Tris-acetate, 1 mM EDTA (pH 8.0)) [8], [9], in the larger lane of the Mupid®-2 Mini-Gel System and running the gel at 100 V for 30–40 min until the bromphenol blue dye
Northern blotting
Total RNA is extracted from the cultured cells with an RNeasy Mini kit (Qiagen), and the concentration is measured by means of a NanoDrop spectrophotometer (NanoDrop Tech). Then 1 to 10 μg of the total RNA is electrophoresed in a 1% agarose gel using the usual TAE buffer without formaldehyde in the large well of the Mupid®-2 Mini-Gel System. Total RNA samples are prepared for loading with 1 ×ばつ formaldehyde gel-running buffer (17.5% formaldehyde and 50% formamide in the final concentration)
Acknowledgements
This study was supported in part by a Research Grant (16A-1) for Nervous and Mental Disorders from the Ministry of Health, Labor and Welfare of Japan.
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