To separate DNA fragments by Agarose Gel Electrophoresis

Objective:- To separate DNA fragments by Agarose Gel Electrophoresis.

Principle:- Nucleic acids are separated by applying an electric field, so these negatively charged

molecules will move through an agarose matrix towards the anode, and the biomolecules are

separated by size in the agarose gel matrix, where the distance travelled by a DNA molecule is

inversely correlated with its size.

Materials:-

i. Agarose powder

ii. 1X TBE buffer (89 mM Tris-base, 89 mM boric acid and 2 mM EDTA) prepared from 10X TBE

iii. Ethidium Bromide (5 mg/ml)

iv. Gel loading dye (Glycerol and orange dye)

v. 1 kb and 100 bp DNA ladder

vi. Horizontal electrophoresis apparatus

vii. Power supply.

Procedure:-

> Measure the desired grams of agarose to make 1% agarose gel.

> Heat the solution to boiling in the microwave to dissolve the agarose to produce a homogeneous mixture.

> Add 4 µl of ethidium bromide CARFULLY to the dissolved agarose and mix.

> Get a gel plate and a comb. Put the two dams into the slots on each side of the gel plate. Make sure that they fit tight. Pour the melted agarose onto the gel plate in the electrophoresis tray.

> Place the comb in its place. Let the gel cool to room temperature. 

> Place the gel in the electrophoresis chamber.

> Pour enough electrophoresis buffer (1X TBE) to cover the gel to prevent overheating of the gel.

> Carefully remove the comb.

> Prepare the DNA sample by mixing around 300 ng of DNA sample with 3-4 µl of loading dye.

> Add 3 µl DNA ladder into the first well by using a micropipette.

> Carefully place the prepared samples into adjacent wells

> Electrophorese the samples at 95 V for 45 minutes. (Check the gel while it is running).

> Carefully remove the gel, place it onto the UV light box and take a picture for the gel. 

Results:- Picture of the gel.