Agarose gel electrophoresis of DNA

Principle:

Agarose gels are more porous and have a larger pore size as compared to polyacrylamide gels and, therefore, used to fractionate larger macromolecules such as DNA or RNA. Porosity of a gel is determined by concentration of agarose – higher the agarose concentration, smaller the pore size and vice versa. When an electric field is applied across the gel, DNA molecules that are negatively charged at neutral pH, migrate towards oppositely charged electrode at rates determined by their molecular size and confirmation. Since charge to mass ratio of NA is constant, the rate of migration is inversely proportional to log₁₀MW, i.e., smaller DNA molecules will travel faster as compared to the larger ones. Further, DNA molecules of the same size but with different conformations travel at different rates. The order of the migration velocity in the increasing order of various forms of DNA is: Supercoiled DNA > linear double stranded DNA > open circular DNA.

Table 1: Percentage of gels and range of resolution for DNA electrophoresis

% Agarose	Range of resolution (linear dsDNA, Kbp)	% Acrylamide	Range of    resolution
			dsDNA (bp)	ssDNA (nt)
0.5	30 – 1.0	3.5	100 – 1000	750 – 2000
0.7	12 – 0.8	5.0	75 – 500	200 – 1000
1.0	10 – 0.5	8.0	50 – 400	50 - 400
1.2	7 – 0.4	12.0	35 – 250
1.5	3 – 0.2	15.0	20 – 150
		20.0	5 - 100

Requirements:

1. 1 X TAE buffer at pH 8.0 (50 X, 24.2 gm Tris, 5.71 ml GAA, 11.1 ml of 0.5 M EDTA, 100 ml DW) Autoclave before use
2. Agarose gel in 1 X TAE
3. Loading dye  (6 X, 6.0 ml of 50% Glycerol, 1.0 ml of 2% BPB, 3 ml sterile DW)
4. Ethidiun Bromide (EtBr)*: 10 mg/ml stock
5. λ/HindIII Marker (23.13, 9.42, 6.56, 2.32, 2.07, 0.56 and 0.13 Kb)
6. Plasmid DNA (pUC18, pBR322)

Procedure:

1.  Prepare 0.8% agarose gel in TAE buffer.

2.  Dissolve agarose completely in micro-oven and cool to 60°C.

3. CAREFULLY, add EtBr into the gel solution to final concentration of 0.5 µg/ml.

4. Pour the molten gel into gel-mould. Immediately position a comb in the mould.

5.  Let the gel cool for 30 minutes.

6.  Pour TAE buffer into the gel buffer reservoirs.

7.  Prepare sample taking 20 µl of DNA sample and mix with 4µl blue juice (6 X).

8.  Carefully remove the comb.

9.  Load the DNA (15 - 20 µl) per well, flanking wells with similarly processed DNA size standard.

Note: the amount of the sample that can be loaded in a well depends on the thickness of the gel as well as dimensions and placing of the comb.

10. Put the lid on the gel apparatus, attach the electrodes and adjust voltage to 100 volts.

11. Allow the gel to run until line of blue juice is visible       near the end of the gel.

12. Turn off the current and visualize the gel in UV - Tran illuminator.

13. Interpret the results.

*Note: EtBr is carcinogen, so, handle with gloves.