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Normalizing cDNA libraries using the EppendorfThermomixer

ology" permits more efficient normalization by accelerating rehybridization conditions in the sample (2). The aqueous phase of the hybridization solution is reduced by adding phenol.

10 g amplified cDNA were precipitated with ethanol and resuspended in 50 l hybrizing buffer with 8% phenol. The phases were mixed by shaking vigorously until a phenol buffer emulsion was formed. This emulsion was maintained by shaking on the Eppendorf Thermomixer. The hybridization reaction was carried out for 20 hours at 37C. A chloroform/isoamyl alcohol extraction with desalination, and selection of the single-stranded DNA took place as described.

Results

The figure shows the result of an agarose gel electrophoresis, which records the changes in the cDNA distribution caused by normalization. Before normalization (lane N0), a series of bands which represent frequently-occurring cDNAs can be seen before a diffuse background of heterogeneous cDNAs. The intensity of these bands decreases after several normalization steps (N1 and N2) have been carried out; conversely, the background of "low copy" cDNAs becomes more intense. This shows the success of normalization, which can be reexamined for cDNAs of frequently-occurring and rare mRNAs with the aid of special probes.

Peter Scheinert & Hans-Joachim Schalk, - Bernhard-Nocht-Institute of Tropical Medicine, Virology Dept.; Hamburg, Germany

Literature

(1) Scheinert, P.; Behrens, B.; Kahle, D.; & Scalk, H.-J. (1996), BIOspektrum 3/96, 50-51. Reprint available: Ref.
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