Universiti Tunku Abdul Rahman (Kampar Campus)

Faculty of Science, Engineering, and Technology

Bachelor of Science (Hons) Biotechnology

Year 2 Semester 1

UESB 2142 Laboratory 2A

(III) Principles of Biotechnology

Lecturer: Dr. Choo Quok Cheong

Student’s Name: Cheah Hong Leong

Student’s ID: 08AIB03788

Experiment No. 4

Title: Restriction Enzyme Digestion

Date: July 21, 2009; August 4, 2009

Title: Restriction Enzyme Digestion

Objective:

– Determine the size of plasmid pBR322 and pGL3X

– Analyzing and reconstructing the plasmid map of pBR322 and pGL3X comparing the

data from single restriction and double restriction that involving EcoRI, PstI, and

SalI.

Materials and Methods:

Refer to Laboratory Manual, UESB 2142 Laboratory 2A (II) Principle of Biotechnology.
Page 22, 23, and 25.
Results:
Part A: Plasmid Mapping of pBR322 (Refer Picture 1 in Attachment)
Table 1: Single and Double Digestion of pBR322
Restriction Enzyme(s) Numbers of Fragments Sizes (kb)
EcoRI 1 ~4.5
PstI 1 ~4.5
SalI 1 ~4.5
EcoRI + PstI 2 ~4.0, ~0.8
EcoRI + SalI 2 ~4.0, ~0.7
PstI + SalI 2 ~3.0, ~1.5

Estimated Size of pBR322: ~4.5 kb

EcoR1
Circularized pBR322 Map: ~0.8 kb
~0.7 kb
S ~1.5 PstI
alI kb

~3.0 kb
Linearized View of pBR322 Map:

E PstI S E
alI
~0.8 ~3.0 kb ~0.7 kb
kb ~1.5
~4.0 kb
kb
~4.0 kb
 ~4.5 kb
Part B: Plasmid Mapping of pGL3X (Refer Picture 2 in Attachment)
Table 2: Single and Double Digestion of pGL3X
Restriction Enzyme(s) Numbers of Fragments Sizes (kb)
EcoRI 1 ~6.0
PstI 1 ~6.0
SalI 2 ~6.0, ~5.0
EcoRI + PstI 1 ~6.0
EcoRI + SalI 2 ~4.0, ~2.5
PstI + SalI 3 ~6.0, ~4.0, ~2.5

Estimated Size of pGL3X: ~6.0 kb

Circularized pGL3X Map: EcoRI
PstI

~2.5 kb

~4.0 kb S
alI

Linearized View of pGL3X Map:

EcoRI S EcoRI
PstI alI PstI

~2.5 kb ~4.0 kb
~6.0 kb

Discussion:
In restriction mapping of pBR322, the estimated size of pBR322 was ~4.5 kb from
the single digestion by EcoRI, PstI and SalI. The theoretical size of pBR322 given was
4.26 kb. The estimated size of pBR322 obtained was still not significantly deviated from
the theoretical size given. However, from the double restriction of EcoRI and PstI; and
EcoRI and SalI, the sizes of pBR322 obtained via summation of the sizes of fragments
were ~4.8 kb and ~4.7 kb respectively, which were more deviated, 0.44 kb to 0.54 kb
from the theoretical value given.

From the single digestion of pBR322 by EcoRI, PstI, and SalI, only one band was
obtained from the gel electrophoresis. This indicates that the three restriction enzymes
were cutting pBR322 only once at one site. From the double restriction of pBR322 by
EcoRI and PstI, only two bands were obtained. EcoRI cutting site was expected to be
~0.8 kb from PstI cutting site. From the double digestion of EcoRI and SalI, two bands
were obtained. EcoRI cutting site was expected to be ~0.7 kb from SalI cutting site. From
the double digestion of PstI and SalI, two bands were also obtained, with sizes of ~3.0 kb
and ~1.5 kb. The shortest distance between the PstI cutting site and SalI cutting site was
expected to be ~1.5 kb. On pBR322, EcoRI cutting site was expected to be laid between
the cutting sites of PstI and SalI with distance of 0.8 kb and 0.7 kb respectively.
The theoretical size of pGL3X given was 6.01 kb. The single digestion of pGL3X
by EcoRI and PstI yielded only one band indicated that EcoRI and PstI cut pGL3X only
at one site. The single digestion of pGL3X by SalI yielded two bands with sizes of ~6.0
kb and ~5.0 kb, in which the sum of the sizes of the bands was ~11.0kb, which was
almost twice the theoretical value given. However, based on Picture 2 in Attachment, the
electrophoresis of undigested pGL3X also yielded single band with size of ~5.0 kb.
Therefore, the band with size of ~5.0 kb from the single digestion by SalI was the
undigested pGL3X due to partial digestion, leaving some pGL3X undigested and remains
circularized. Hence, SalI was also expected to have only one cutting site on pGL3X.
The double digestion of EcoRI and PstI only yielded one band in electrophoresis
with size of ~6.0 kb. One possible reason for this result was the one of these restriction
enzymes did not have any cutting site on pGL3X, but this possibility was rejected by the
single digestion results which proved that both EcoRI and PstI have single cutting site on
pGL3X. Another possible reason for this result was the cutting site of the two restriction
enzymes were either too close to each other with shortest distance of may be only few or
several base pairs, or there was overlapping of recognition and cutting site of the two
restriction enzymes. However, the second possible reason was unlikely as both EcoRI
and PstI are unambiguous with restriction site of 5’-G! AATTC-3’ and 5’-CTGCA! G-3’,
respectively.

Double digestion of pGL3X by EcoRI and SalI yielded two bands with sizes of
~4.0 kb and ~2.5 kb. The sum of the sizes of the bands was ~6.5 kb, which was little
deviated from the theoretical size of 6.01 kb given. This indicated that EcoRI and SalI cut
pGL3X at two different sites with shortest distance between the two cutting sites was
~2.5 kb.
Suppose from the results of double digestion of pGL3X by EcoRI and PstI; and
EcoRI and SalI, it was expected that the double digestion by PstI and SalI yielded only
two bands with sizes of ~4.0 kb and ~2.5 kb. However, the electrophoresis yielded three
bands for Well 8 with double digestion by PstI and SalI, with sizes of ~4.0 kb, ~2.5 kb,
and another band with size of ~6.0 kb. The ~6.0 kb band was the linearized pGL3X due
to partial digestion, leaving some pGL3X was only cut by either PstI or SalI.

Conclusion:

EcoR
pBR322 Map: 1 ~0.8 kb
~0.7 kb
S ~1.5 PstI
alI kb

~3.0 kb

pGL3X Map: EcoRI

PstI

~2.5 kb

References: ~4.0 kb S
Brown, T. A. (2006). Gene Cloning & DNA Analysis: AnalIIntroduction, 5th ed., Oxford,
U.K: Balckwell Publishing Ltd.
Dale, J. W. & Schantz, M.V. (2007). From Genes to Genomes: Concepts and
Applications of DNA Technology, 2nd ed., West Sussex, U.K: John Wiley & Sons Ltd.
Restriction Enzyme. (2009, August 4). In Wikipedia, the free encyclopedia. Retrieved
August 15, 2009, from http://en.wikipedia.org/wiki/Restriction_enzyme

Attachment:

1 2 3 4 5 6 7 8
~4500 bp
 >10000 bp

~6000 bp
~4000 bp
~3000 bp ~3000 bp

~1500 bp
~800 bp

~700 bp

Picture 1: Gel electrophoresis result on pBR322 and DNA marker (1kb Blue DNA
Ladder)
Note:
Well 1: DNA Marker (1kb Blue DNA Ladder)
Well 2: Undigested pBR322
Well 3: pBR322 + EcoRI
Well 4: pBR322 + PstI
Well 5: pBR322 + SalI
Well 6: pBR322 + EcoRI + PstI
Well 7: pBR322 + EcoRI + SalI
Well 8: pBR322 + PstI + SalI

1 2 3 4 5 6 7 8
 ~6000 bp
~5000 ~4000 bp
bp
~2500 bp

Picture 2: Gel electrophoresis result on pGL3X and DNA marker (1kb Blue DNA
Ladder)
Well 1: : DNA Marker (1kb Blue DNA Ladder)
Well 2: Undigested pGL3X
Well 3: pGL3X + EcoRI
Well 4: pGL3X + PstI
Well 5: pGL3X + SalI
Well 6: pGL3X + EcoRI + PstI
Well 7: pGL3X + EcoRI + SalI
Well 8: pGL3X + PstI + SalI