Molecular Waight Marker & Loading Dye

Be careful to run a molecular weight marker.

To monitor electrophoresis in agarose gel, molecular weight marker is usually loaded with conc dye. For an instance, sample lanes contain 1uL of sample, 1uL of loading dye and 8uL of water, while the lane for marker does 1uL of the molecular weight marker and 9uL of the dye. If you wish to take a photo of gel of publication quality, be careful. Bands of a lane containing conc dye may be broad.

Simple and easy loading dye.

Loading dye containing xylene-cyanol is light and sticky and is difficult to be applied. Also, it is sometimes inconvenient to take photo because of the light blue color superimposed onto DNA bands. It is better to use loading dye containing only bromo-phenol-blue (BPB) for marker. I usually make two batches of loading dye containing regular (c.a.0.1%) and conc (c.a.1%) BPB. Then I mix them to make appropriate blue color. It is difficult to weigh BPB precisely sticking to a balance dish. Sucrose is better than glycerol for increasing density, because scrose is less sticky. SDS is sometimes added to stop enzyme reaction. Loading sample containing SDS is however, sticky to the pipette tip or onto parafilm and is difficult to handle. Adding high concentration (7M) of urea instead of SDS makes the loading dye much easier to handle. To kill nuclease, adding EDTA-3Na is convenient, because this neutralizes the dye (c.a.pH7-7.5) without addition of tris-base.

Recipe for loading dye for 40mL

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Sucrose ----- 20g
Urea -------- 16.82g
0.5M EDTA --- 0.8mL
BPB --------- 40mg(0.1%) - 400mg(1%)

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Add small amount of water to 40mL.
Autoclaved and stored at room temperature.

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