Agarose gel electrophoresis

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Agarose gel electrophoresisAgarose gel electrophoresis protocol (principles, materials and procedures):

Principles: Agarose rarely interact with biomolecules and has been used in seperating proteins and nucleic acids. When heated argarose solution becomes gel with pore size from 50nm to 200nm. The migeration rates are determined by the length of the DNA, voltage fo the electrophoresis and the concentrration of the agarose. With addition of a fluorescent dye like EB or gold view, DNA can be seen under an UV detector.

Materials: DNA electrophoresis apparatus, pipet, loading tip, 10X loading buffer, DNA marker (ladder)

agarose concentration versus optical range of DNA size :

Agarose concentration(%)

0.3

0.6

0.7

0.9

1.2

1.5

2.0

DNA(Kb)

5~60

1~20

0.8~10

0.5~7

0.9~6

0.2~3

0.1~2

Running buffer: 0.5X TBE: 5.4 g of Tris base, 2.75 g of boric acid, 2 ml of 0.5 M EDTA in 1 liter water solution (pH=8.0)

or                    1X TAE: 4.84g of Tris base, 1.14ml of glacial acetic acid, 2ml of 0.5M EDTA  in 1 liter water solution (pH=8.0)

The agarose solvent should be the same with the running buffer.

Agarose Gel Protocol:

1. Pour enough running buffer into the electrophoresis tank. (The surface should be higher than the top of the gel and not overflow)

2. Prepare the suitable concentration of agarose solution and microwave it untill a boiling. Add in 5-10µl of gold view dye when it's cooled down to 55°C

3. Choose the suitable gel tank, pour the fluid agarose gel and insert the comb

4. After a complete solidification put the agarose gel in the electrophoresis tank

5. Mix the sample with loading buffer sufficiently and load them into the sample lane together with the marker (usually marker in the first lane).

6. Set an appropriate voltage and run the electrophoresis.

7. After appoximately 35min (80V), 25min (100V),  put the agarose gel in an UV detecor and record the picture.