Describe the main purpose of DNA electrophoresis. What are the two main ingredie

Question

Describe the main purpose of DNA electrophoresis.

What are the two main ingredients that are used to make a DNA gel?

Describe the composition and purpose of the buffer.

What is added to the DNA samples to help visualize them while loading into the gel wells?

During electrophoresis, what sign will you see in the buffer that will tell you that electric current is running?

What stain is used to see the DNA when the gel is exposed to UV light?

Describe the pattern of migration of DNA of different sizes.

There are 3 major DNA sizes in your sample. What are the sizes, in bases, of the three DNA fragments?

Explanation / Answer

1. DNA electrophoresis is an analytical technique used to separate DNA fragments by size (molecular weight).

2 & 3. The main ingredients to make an electrophoresis gel (there is nothing like DNA gel) is Agarose and buffer. Agarose is purified form of Agar. Agar is composed of sulfated galactose. The sulfate groups are removed (that is, agarose is highly purfied form) from Agar so as to make it neutral and not interfere with migration of charged particles, it is then called Agarose. The buffer most commonly used is either TAE or TBE (tris acetate EDTA) or (Tris borate EDTA). The buffer imparts negative charges on the DNA molecules, chelates all Mg2+cations required for DNase activity, an enzyme that may degrade DNA.

TAE buffer is prepared by addid 24.2 grams Tris base in water, 5.71 ml galcial acetic acid, 10 ml of 500mM EDTA at pH 8.0 (EDTA wont dissolve if the pH is below 8), and the final volume is made to 100 ml. This stock solution can be diluted 50:1 with water to make working solution.

4. The loading buffer is used to DNA samples while loading. The buffer is made in glycerol, along with xylene cyanol and bromophenol blue. Glycerol imparts density and ensures that the sample settles down and do not float in the buffer.

5. To check if the electric current is running or not, check the electrode wires, bubbles come out at the electrode wires, which is an indication of electric current running.

6. Ethidium bromide, an intercalating agent of DNA is added to visualize them.

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