Gel Electrophoresis of DNA

I learned how to do DNA Extraction during the last week. I used Agarose Gel Electrophoresis technique to for the separation of Agrobacteria’s DNA fragments. The result of Gel Electrophoresis is the separation of charged molecules. DNA is a negatively charged molecule, and is moved by electric current through a matrix of agarose.

Electrophoresis is a method used in the laboratory that results in the separation of charged molecules. DNA is a negatively charged molecule, and is moved by electric current through a matrix of agarose.

 At first, I made 1% Agarose which was contain 1 gram molecular Biology Agarose and100 ml 1x Tris-acetate-EDTA (TAE) buffer. After mixing Agarose powder with TAE buffer, I heated my solution in a microwave oven until completely melted.

Then, I poured hot Agarose solution into a casting tray and placed a sample comb into the top part of tray and allowed Agarose to cool down and solidify at room temperature. Meanwhile, I tried to make my DNA samples. I set up 1 Ruler and 4 samples for Agrobacteria’s DNA.

DNA Samples
Agrobacteria's DNA	7 µL
5x Orange G Loading Dye	2 µL
Sybergreen	1 µL

Ruler
PCR MW Ruler	9 µL
Sybergreen	1 µL

 When the Agarose was solidified, I lifted up the comb gently. The comb was created several places on the gel to load different samples.

I poured TAE buffer into the gel running tank and it should cover gel surface. Then, I tried to load my samples. I loaded 10 µL of the Ruler with a Micropipettor into first well from left side. Then, I started to load 10 µL of four Agrobacteria’s DNA samples with a Micropipettor into each well of my gel.

When my gel was prepared and loaded, the electrical leads on the running tank were connected to a power supply. I connected the leads in the way that the red (positive) lead is at the end of the gel to which the DNA will migrate and the black (negative) lead is at the end of the gel containing the wells. Then, I set power supply on 120 V and allowed my gel to run for 60 minutes and I placed a bucked on top of the gel running tank because the gel has to run in the dark.

After 60 minutes, I turned off the power supply and unplugged the gel running tank. Then, I removed our gel from the running tank and carefully carried the gel to the ultraviolet light-box. Because gel holder was not UV transparent so I placed the gel onto the glass surface of the light-box. The DNA bands in the gel light up when exposed to UV light. The box exposes the gel to UV light. I took a picture from my gel.