Scribd
Upload
202 views3 pages

Ecor1 (0/4363) : 18 April 2023

This document provides instructions for an assignment on restriction mapping of plasmid pBR322. Students are asked to conduct restriction digests on pBR322 using various restriction enzymes. They will separate the resulting fragments by agarose gel electrophoresis and use the results to build a physical map of the restriction sites. The document includes a proposed restriction map of pBR322 showing sites for the enzymes Pst1, EcoR1, and Hind111. It also provides sample data from restriction digests and gel electrophoresis for students to analyze.

Uploaded by

Thandeka Nsele
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
202 views3 pages

Ecor1 (0/4363) : 18 April 2023

This document provides instructions for an assignment on restriction mapping of plasmid pBR322. Students are asked to conduct restriction digests on pBR322 using various restriction enzymes. They will separate the resulting fragments by agarose gel electrophoresis and use the results to build a physical map of the restriction sites. The document includes a proposed restriction map of pBR322 showing sites for the enzymes Pst1, EcoR1, and Hind111. It also provides sample data from restriction digests and gel electrophoresis for students to analyze.

Uploaded by

Thandeka Nsele
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
You are on page 1/ 3

Bioc 315 Assignment: Restriction Mapping 18 April 2023

Name: …………………………………………………………………… Student No. ……………………………………

RESTRICTION ANALYSIS OF PLASMID pBR322


In this exercise, several restriction endonuclease digestions will be carried out on plasmid DNA, the
resulting restriction fragments will be separated by electrophoresis on an agarose gel, and from the results
you will build a “physical map” indicating where the restriction endonuclease cleavage sites lie relative to
one another on the plasmid DNA molecule.

EcoR1 (0/4363)

Pst1 (3613)

Hind111 (29)
pBR322

(4363bp)

Fig1: Restriction Map of Plasmid pBR 322 showing the location and positions of restriction sites – Pst1,
EcoR1 and Hind111

Figure 1: Agarose (1%) Gel electrophoresis of pBR 322 restricted with several restriction enzymes and
combinations of them.
Lane 1: Uncut pBR322.
Lane 2: 1KB Ladder.
Lanes 3, 4 & 5: Restriction digests of pBR 322 with Restriction enzymes Pst1, EcoR1 and Hind 111
respectively; 4363bp.
Lane 6: Restriction digests of pBR 322 with Pst1 & EcoR1; 3613 & 750 bp respectively
Lane 7: Restriction digests of pBR 322 with Pst1 & Hind111; 3584 779 bp respectively
Lane 8: Restriction digests of pBR 322 with EcoR1 & Hind111; 4334 & 29bp respectively.

1 Analysis of Results [Refer to figures 1 and 2]


1. Is the original molecule Linear or Circular? Explain. [3]
2. What are the two forms of DNA represented in Lane 1, and why? [4]
3. What is a DNA Ladder mix, and what purpose does it serve? [3]
4. Why is there only 1 band resolved in Lane 8? [2]
5. Give the specific Base sequences recognized by Eco R1 and Hind III [4]
6. Electrophoresis of DNA is carried out at neutral pH (7.5). Why? [2]
7. Why is Ethidium bromide the stain of choice for DNA gels? [4]

2. A 10 kb Linear DNA Molecule was cleaved with several Restriction Enzymes. The size of each fragment
was determined by agarose gel electrophoresis.

TABLE 1: Restriction enzymes and fragments generated.

Enzyme Fragment size


Hind 111 4.5kB 5.5kB
Bgl 1 6.2kB 3.8kB
Hind 111 / Bgl 1 4.5kB 1.7kB 3.8kB

2.1 Is the original molecule linear or circular? Why? [2]

2.2 Draw a map of restriction sites showing distances between sites that are consistent with the data
presented [6]
3 Below is a restriction map for the plasmid pGEN101 (total length = 20 kb). Using this map as a guide, give
the number of restriction fragments along with their associated lengths that would result from digesting
pGEN101 with the restriction enzymes EcoRI, BamHI, and a combination of EcoRI + BamHI.

6kb
BamHI
BamHI
2kb

BamHI

4kb
8kb

EcoRI

What are the sizes of the fragments generated by:-

3.1 Eco R1
3.2 Bam H1
3.3. Eco R1 + Bam H1

4. A DNA molecule was cleaved with several restriction enzymes or combinations of enzymes. The size of
each fragment was determined by agarose gel electrophoresis. The following data were obtained.

TABLE 1: Restriction enzymes and fragments generated

Enzyme Fragment size


Pst 1 1.9kB, 0.7kB
Bam H1 1 x 2.6kB
Hind III 1 x 2.6kB
Pst 1/ Bam H1 0.7kB, 0.9kB, 1.0kB
Pst 1/ Hind III 1.9kB, 0.3kB, 0.4kB
Bam H1/ Hind III 1.2kB, 1.4kB

4.1 Is the original molecule linear or circular [2]

4.2 Draw a map of restriction sites showing distances between sites that are consistent with the data
presented [8]

Resources to assist with your assignment


https://www.youtube.com/watch?v=vq759wKCCUQ
https://www.youtube.com/watch?v=ZXdrVl0ywOg&feature=youtu.be
https://www.youtube.com/watch?v=kdjgZiFwm_k
https://www.youtube.com/watch?v=v2T8Y3-8674&t=151s
https://www.youtube.com/watch?v=vq759wKCCUQ

You might also like