Visualization of a low concentration and molecular weight DNA (DNA gel stain)
Stephane Fadanka, Nadine Mowoh, Mujar Minette Shalo
Abstract
After PCR amplification of DNA, agarose gel electrophoresis is run to separate the DNA based on their size.
The agarose gel consists of microscopic pores that act as a molecular sieve which separates molecules based on their charge, size and shape. Agarose gel electrophoresis can also be used to separate other charged biomolecules such as RNA and proteins. Agarose is isolated from the seaweed genera Gelidium and Gracilaria and consists of repeated agarobiose (L- and D-galactose) subunits. The concentration of agarose in a gel depends on the sizes of the DNA fragments to be separated, with most gels ranging between 0.5%-2%.
This protocol describes the use of BenBio DNA gel stain for the visualization of low molecular weight and low concentration DNA with agarose gel electrophoresis.
The BenBio DNA gel stain is therefore, a good alternative to EtBr-based DNA gel stains.
Before start
Ensure all materials, equipment and chemicals to be used for this experiment are all in place before starting.
Steps
Visualization of a low concentration and molecular weight DNA
To confirm visualization of a low molecular weight DNA
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Follow the steps in preparing a 2% agarose gel as described in this protocol,
NoteWe use the TO-DMSO DNA gel stain as "test DNA gel stain" and an EtBr based DNA gel stain as "positive control or standard DNA gel stain" (meaning 2 gels will be prepared and run simultaneously, where possible. If this is not possible, run one gel to finish and run the next. -
Load the gel (made from the test gel stain) by pipetting 3 to 5 uL of sample into the wells ( in this case a loading dye would not be used since the amplicon/ sample is a DNA ladder that is made with the tracking dye in it.
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Load the second gel (made from the control gel stain) by pipetting 3 to 5 uL of sample into the wells ( in this case a loading dye would not be used since the amplicon/ sample is a DNA ladder that is made with the tracking dye in it).
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Connect the electrophoresis units to a power pack and run the 2 gels simultaneously under the same conditi ons (when using TBE running buffer, we run at 80 to 100 Volts for 15 to 20 minutes) to finish.
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Transfer the gel unto a UV transilluminator and visualize the resulting band separation to confirm separation of the ladder into visible distinct bands (from the lowest to highest base pair bands).
To confirm visualization of a low concentration DNA
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Make 1:5 serial dilutions of the 100bp DNA ladder or any DNA amplicon and use the different dilutions as amplicons to load a gel well.
Note2 gels would be prepared, one with the test gel stain and another with the control gel stain.These gels could be run simultaneously or one after the other. -
Repeat the steps described above in loading a gel using the different dilutions of the DNA.
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Connect the electrophoretic unit to the power source and allow the gel to run through (when using TBE buffer, we run at 80 to 100 Volts for 15 to 20 minutes).
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Transfer the gel unto a UV transilluminator and visualize to identify visible, sharp bands which determine the highest dilution of the DNA that is detectable by the gel stain.
