Question 1

What is agarose and why is it used in gel electrophoresis?

Accepted Answer

Agarose is a polysaccharide derived from red algae that forms a porous matrix when dissolved in buffer and solidified. The hydrogen-bonded agarose molecules create pores through which DNA molecules migrate during electrophoresis. Different agarose concentrations produce varying pore sizes, allowing separation of DNA fragments by size.

Question 2

How does the loading buffer help prepare DNA samples for gel electrophoresis?

Accepted Answer

The loading buffer contains glycerol, which increases sample density and helps DNA settle at the bottom of wells. It also includes tracking dyes like xylene cyanol and bromophenol blue that monitor the migration progress of DNA bands through the gel during electrophoresis.

Question 3

Why do smaller DNA molecules move faster through the agarose gel than larger ones?

Accepted Answer

Smaller DNA molecules navigate through the agarose pores more easily than larger molecules because they encounter less resistance. When an electric potential is applied, negatively charged DNA migrates toward the positive electrode, with size determining migration speed and allowing separation by molecular weight.

Question 4

What role does ethidium bromide play in DNA visualization?

Accepted Answer

Ethidium bromide is a fluorescent dye added to the agarose solution that intercalates between DNA bases. Under UV light, the stained DNA molecules fluoresce as visible bands, enabling researchers to identify and locate separated DNA fragments on the gel for analysis.

Question 5

How is gel extraction used to purify DNA fragments after electrophoresis?

Accepted Answer

After separating DNA fragments by electrophoresis, individual bands are isolated and cut from the gel. The gel slice is then processed through a commercial extraction kit where agarose is dissolved, DNA binds to an anionic resin column, impurities are washed away, and pure DNA is eluted with buffer or deionized water.

Question 6

Why are lower agarose concentrations and lower voltages used for gel extraction protocols?

Accepted Answer

Lower agarose concentrations (0.7-0.8%) ensure efficient migration of DNA bands, while lower voltage prevents gel heating and DNA damage. A wide-combed gel cast produces thick, easily isolated bands. Short UV exposure during band visualization also minimizes DNA damage during the extraction process.

Question 7

How do researchers determine the size of unknown DNA fragments on a gel?

Accepted Answer

A DNA ladder containing fragments of known sizes is run alongside unknown samples on the same gel. The position of unknown DNA bands is compared with the ladder's reference bands under UV light, allowing researchers to estimate the molecular weight of their DNA fragments of interest.

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