Exp 4 Restriction Enzyme

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) EcoRI PstI SalI EcoRI + PstI EcoRI + SalI PstI + SalI

Numbers of Fragments 1 1 1 2 2 2

Sizes (kb) ~4.5 ~4.5 ~4.5 ~4.0, ~0.8 ~4.0, ~0.7 ~3.0, ~1.5

Estimated Size of pBR322: ~4.5 kb

Circularized pBR322 Map: ~0.7 kb

EcoR1

~0.8 kb

~1.5 kb

S alI

PstI

~3.0 kb Linearized View of pBR322 Map:

E

PstI

~0.8 kb

S alI

~3.0 kb

E

~0.7 kb

~4.0 kb ~4.0 kb

~1.5 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) EcoRI PstI SalI EcoRI + PstI EcoRI + SalI PstI + SalI

Numbers of Fragments 1 1 2 1 2 3

Sizes (kb) ~6.0 ~6.0 ~6.0, ~5.0 ~6.0 ~4.0, ~2.5 ~6.0, ~4.0, ~2.5

Estimated Size of pGL3X: ~6.0 kb Circularized pGL3X Map:

EcoRI PstI

~2.5 kb

S alI

~4.0 kb Linearized View of pGL3X Map: EcoRI PstI

EcoRI PstI

S alI

~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:

pBR322 Map:

~0.7 kb S alI

EcoR 1

~1.5 kb

~0.8 kb PstI

~3.0 kb pGL3X Map:

EcoRI PstI

~2.5 kb

References:

S ~4.0 kb 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 bp

~4000 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