Lab Report 2

Kan Karin

14/9/2016

Mapping DNA using Restriction Enzymes and

Electrophoresis
Summary
Mapping DNA was performed by groups formed by two students in this
laboratory session. The mapping is done with the aid of restriction
enzymes (RE). In this laboratory session, unknown restriction enzymes
and lambda DNA is given. By electrophoresis and comparing the data
collected to the given restriction map of lambda DNA, the unknown
restriction enzymes are identified.

Introduction
Restriction enzymes are found in bacteria and are able to recognize
certain DNA sequences, known as restriction site, and cleave it. They
protect the bacteria from viral infection by cleaving the invading viral
DNA. Different restriction enzymes work at different conditions. Variables
include reaction buffer, temperature for incubation.
If two kinds of restriction enzyme (eg. A, B) are present to digest the DNA
template simultaneously - double digestion, the DNA sequences specified
by either of them will both be cut. By electrophoresis and comparing the
length of DNA fragments in double digestion to the that in single digestion
(only one kind of restriction enzyme digest the DNA template), we can
generate a restriction map. The relative locations of restriction site with
respect to one another in the DNA molecules can then be determined.
In this lab session, each group is given with 4 tubes containing different
restriction enzymes (RE) but the same DNA template (lambda DNA). The
first 3 tube (tube A, B, C) contain Hind III, RE B and RE C respectively, in
which single digestion was carried out. The fourth tube (tube D) contains
RE C and RE D, in which double digestion was carried out. After the
digestion, we will let the mixture in tubes undergo agarose gel
electrophoresis.
Tube A, containing Hind III, is for construction of standard curve. The
standard curve plots the size of DNA fragment (in kb) against the distance
it migrates during electrophoresis. With the aid of standard curve, we can
determine the size of the fragment from the gel photo by measuring how
far the DNA fragments have migrated.
Higher concentrations of agarose in the electrophoresis allow separation
of small DNA while lower concentrations of agarose facilitate resolution of
larger DNA. In this experiment, low concentrations of agarose (0.8%) is
chosen. Moreover, since EtBr is carcinogenic, Gel Red solution is used as
substitute to visualize the DNA fragment bands.

Lab Report 2

Kan Karin

14/9/2016

Materials and Methods

Please refer to the lab manual with amendments on the amount of lambda
DNA added. Instead of 1.25l, 1.0l lambda DNA is added to each tube.
Accordingly, The amount of water added to tube A, B, C is changed from
6.75l to 7.0l. The amount of water added to tube D is changed from
5.75l to 6.0l.

Results and discussions

By matching the band to the the restriction map, the sizes of the
fragments produced by Hind III are found.
Fragmen
t size
554
2027
2322
4361
6682
9416
(kb)
Distance
travelled
4.95
3.5
3.35
2.7
2.35
1.85
by band
(cm)
Table 1. Distance travelled by fragments of different sizes in tube A
A standard curve is plotted and attached.
The result of tube B and C are as follow. The sizes of fragments are found
from the standard curve.
Distance
travelled by
5.45
4.95
band (cm)
The
corresponding
350
560
size of
fragments (kb)
Table 2. Sizes of fragments in tube B
Distance travelled by
band (cm)

5.1

3.7

3.6

3.35

1.80

2500

11000

3.35

3.25

2.95

Lab Report 2

Kan Karin

The corresponding
size of fragments
510
1800
(kb)
Table 2. Sizes of fragments in tube C

14/9/2016

200
0

2600

280
0

380
0

940
0

After comparing the sizes of fragments to the given restriction map of

lambda DNA, it is believed that RE B and RE C is Bgl II and Noe I
respectively.
As for tube D, the distance travelled by the bands is measured and the
fragments sizes are found from the standard curve.
Distance
travelled
by band
(cm)
The
correspond
ing size of
fragments
(kb)

2.6
5

2.9
5

3.3

3.4

3.6

3.7

3.8

4.3
5

4.5
5

4.9
5

500
0

380
0

260
0

240
0

200
0

180
0

1650

96
0

800

540

After comparing the sizes of fragments to the given restriction map of

lambda DNA, the most possible combination of restriction enzyme in tube
D is Noe I and EcoR I, although one of the band (fragments of 2600kb)
fails to match the fragments produced in Noe I-EcoR double digestion.

Lab Report 2

Kan Karin

14/9/2016

References
Jorgensen, R. A., Rothstein, S. J., & Reznikoff, W. S. (1979). A restriction enzyme cleavage map of
Tn5 and location of a region encoding neomycin resistance. Molecular and General Genetics
MGG, 177(1), 65-72.
Morrow, J. F., & Berg, P. (1972). Cleavage of Simian virus 40 DNA at a unique site by a bacterial
restriction enzyme. Proceedings of the National Academy of Sciences, 69(11), 3365-3369.
Smith, H. O., & Birnstiel, M. L. (1976). A simple method for DNA restriction site mapping. Nucleic
acids research, 3(9), 2387-2398.