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
|
