Electrophoretic separation of macromolecules is a commonly employed method in molecular biology. Knowing the dispensed-sample quantity is decisive for reliable interpretation of the bands.
Depending on the kind of molecules to be separated, polyacrylamide or agarose is often used as matrix.
The various available methods exploit the migration of the molecules in the electric field on the basis of their electric charge and molecular weight. After separation, the molecules are usually fixed and stained.
Visual detection limits are:
- Proteins – Coomassie Brilliant Blue R-250: 50–100ng per band; Silver staining: 5–10ng per band.
- DNA:Ethidium bromide staining: approximately 50ng per band.
The correlation between the first step of the electrophoresis and the final staining is often ignored.
Whether a band lies above or below the visual detection limit depends primarily on the amount of dispensed sample. Accurate determination of the sample quantity dispensed with a micropipette is often difficult, because, on the one hand, the amount of aspirated liquid may differ from the pipette setting and, on the other hand, the sample quantity actually delivered to the gel can differ from the nominally set quantity due to casting tolerances or damage of the gel pockets.
GLP guidelines stipulate, among other instructions, that all raw data collected during the execution of a test must be recorded directly, exactly and legibly. The dispensed amount of liquid, which is to be documented exactly according to the GLP guidelines, is decisive for the subsequent analysis of the gels.
The Transferpette electronic here provides a solution. As with other electronic pipettes, a touch of the pipetting button suffices to pick up the sample. Unlike other pipettes however, the GEL mode (patent pending) of the Transferpette electronic enables the user to control and document the actual volume aspirated and dispensed during the pipetting operation.
For example, if the pipetting button is pressed a second time again during the sample aspiration process, aspiration stops and the display shows the exact amount of sample acquired. When the pipetting button is pressed again, the pipette dispenses the liquid very slowly, at a fixed, uniform speed to avoid the creation of eddies.
When the pocket in the gel is full and the pipetting button is pressed once again, the dispensing stops and the display shows the exact amount of sample dispensed. This is decisive information for accurate interpretation of the electrophoresis results if the dispensed volume is less than the aspirated volume.
The displayed volume on the Transferpette electronic may then be used in documentation of the test for GLP purposes. In turn, the pattern of the bands can be interpreted correctly, since the concentration of the dispensed sample and the dispensed amount of sample are known exactly.
Dr Antonio Romaguera, is with Brand GmbH + Co KG, Wertheim, Germany. www.brand.de
