Question

In genetics when using gel electrophoresis there are many decisions. If you want to separate fragments that are very close in size you should use a higher percentage of agarose. The power supply has two settings for voltage, 120V or 160V. It also has a timer that has 2 settings 15 minutes and 90 minutes. If you would be running whole genomic DNA samples you recently extracted and the fragments would be about 30 kb in size. If  performed a number of PCR reactions that should produce fragments of 450 bp. You generally would not run the genomic DNA and the PCR products on the same gel due to the difference in their sizes.

- What settings would you use for genomic DNA gel vs the gel with the PCR fragments?

- Which gel the genomic or PCR products would you add ethidium bromide to and which gel would you stain in an ethidium bromide bath? Explain why.

Answer 1

Gel electrophoresis is a technique use to separate DNA fragments according to their size. In gel electrophoresis, charged molecules are placed in an electrical field and allowed to migrate towards the positive and negative poles. The molecules separate because they move at different rates due to their differences in charge and size. Because DNA is negatively charged, it is loaded into wells at the negative pole of the gel and migrates towards the positive.

Each fragment's migration rate is dependent on the molecular weight of the fragment. Smaller fragments molecules move faster than larger molecules through the gel. Higher concentrations of gel material provide better resolution of small fragments and lower concentration of gel material provide better resolution to larger fragments

Agarose gel are usually uesd to separate DNA fragments electrophoretically. Higher concentrations of Agarose provide better resolution to smaller fragments and vice versa.

A) * The genomic DNA of size 30 kb size - 30 kb is large DNA fragment so it requires lower agarose concentration. 0.5% agarose is used to make gel.

* The DNA fragments produced after PCR is of 450bp - As the fragment size is small, higher concentrations of Agarose is used. 1.2% Agarose is used to make gel.

B) In agarose gel electrophoresis the gel is placed in electrophoresis tank, cover with buffer, and sample are loaded by injecting the sample into the wells. Once the system runs, the DNA in the gel need to be stained and visualised . Ethidium bromide  is reagent used in agarose gel electrophoresis . Ethidium bromide is an intercalating agent( fluorescent dye) . Ethidium bromide is a cyclic planar molecule that binds between the stacked base pairs of DNA. The ethidium bromide bind with DNA and under ultraviolet light the DNA bands fluoresce orange- red colour.

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