DNA ELECTROPHORESIS:

DNA ELECTROPHORESIS

1 KB Ladder DNA:

Description: A number of proprietary plasmids are digested to completion with appropriate restriction enzymes to yield 10 bands suitable for use as molecular weight standards for agarose gel electrophoresis. The digested DNA includes fragments ranging from 0.5 to 10.0 Kb. The 3.0 Kb fragment has increased intensity to serve as a reference band.

Source: dsDNA is digested to completion with appropriate restriction enzyme; phenol extracted and equilibrated to 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA.

Concentration: 500 µg/ml

Storage condition: 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. For long term storage, store at -20°C. DNA ladders are stable for at least three months at 4°C.

Notes: All fragments have 4-base 5’-overhangs that can be labeled using T4 Polynucleotide Kinase or filled-in using DNA polymerase I Klenow fragment. Use α-[32P] dATP or α-[32P] dTTP for fill-in reaction. Dilute in TE or other buffer of minimal ionic strength. DNA may denature if diluted in dH₂O and run aberrantly in gel.

Photograph 1: Horizontal Electrophoresis set running DNA on Agarose gel 

Usage recommendation: Load 0.5 µg of the 1 KB ladder diluted in sample buffer. The 1 KB ladder was not digested for precise quantification of DNA mass, but can be used for approximating the mass of DNA in comparably intense samples of similar size. The approximate mass of DNA in each of the bands is given below (assuming a 0.5 µg loading).

Fragment	Base pairs (bp)	DNA mass (ng)
1	10002	42
2	8001	42
3	6001	50
4	5001	42
5	4001	33
6	3001	125
7	2000	48
8	1500	36
9	1000	42
10a	517	42
10b	500

Figure 1: Molecular size of 1 Kb ladder (DNA marker)

Source: A practical manual for molecular biology. Tiwari KB

Photograph 2: DNA stained with EtBr under UV illumination

Amount of agarose gel and efficient range of separation of linear DNA molecules

Amount of Agarose in Gel (%)	Efficient range of separation of linear DNA molecules (Kb)
0.3	60-5
0.6	20-1
0.7	10-0.8
0.9	7-0.5
1.2	6-0.4
1.5	4-0.2
2.0	3-0.1