Università degli Studi di Urbino Carlo Bo / Portale Web di Ateneo


LABORATORY OF BIOTECHNOLOGY II
LABORATORIO DI BIOTECNOLOGIE II

Techniques of gene cloning into a prokaryotic expression vector
Tecniche di clonaggio genico in un vettore di espressione procariotico

A.Y. Credits
2015/2016 8
Lecturer Email Office hours for students
Marzia Bianchi

Assigned to the Degree Course

Giorno Orario Aula

Learning Objectives

The goal of this course is to give the basic theoretical notions and practical skills of the most popular DNA cloning techniques used to investigate the structure and function of genes (from DNA to protein). At the end of the course the students are expected to acquire a good knowledge of the recombinant DNA technology and to develop skills in designing and performing a cloning experiment autonomously.

Program

Laboratory experiences
1. Defining the goal of the cloning experiment and design of the cloning strategy.
1.1 Selection of gene to be cloned and cloning vector.
1.2 Selection of restriction enzymes to be used and design of degenerate primers.
2. Isolation of total RNA from the selected source.
2.1 Spectrophotometric quantification and electrophoresis of purified RNA.
3. cDNA synthesis from total RNA, by reverse transcription (RT).
4. PCR with degenerate primers specific for the insert to be cloned.
4.1 Analysis of the amplified product by agarose gel electrophoresis.
4.2 Purification of the PCR product.
5. Ligase of the PCR product with a plasmid vector, by the TA cloning strategy.
6. Transformation of competent E. coli cells with the ligase reaction.
7. Screening and identification of recombinant clones.
7.1 PCR from bacterial colonies.
7.2. Extraction of one positive recombinant clone (plasmid miniprep).
8. Transfer of the target gene into a prokaryotic expression vector, by directional cloning, with restriction enzymes.
8.1 Restriction enzyme digestion of both insert and vector.
8.2 Ligase reaction.
8.3 Transformation of a competent cloning strain.
8.4 Screening of recombinants.
9. Transformation of the expression construct into a host strain, suitable for recombinant protein expression.
9.1 Screening, propagation and storage of recombinant clones.


Lectures (supporting practical experiences)
1. DNA cloning: overview of enzymes, vectors, host cells.
1.1 Enzymes for DNA cloning: nucleases, ligases, polymerases, phosphatases.
1.2 Cloning vectors for E. coli and eukaryotic cells: plasmids, phages, cosmids, BAC, YAC, shuttle vectors.
1.3 Biological systems of molecular biotechnology: bacteria, yeast; higher eukaryotes.
2.DNA introduction into live cells.
2.1 Genetic transformation of prokaryotes.
3. Construction and screening of DNA libraries.
3.1 Preparation of genomic DNA and cDNA for library construction.
3.2 Library screening strategies.
3.3. Colony and plaque hybridization: production of labeled probes.
3.4 Isolation of recombinant vectors and restriction mapping and/or sequencing
4. Polymerase chain reaction (PCR) as an alternative to cell-based DNA cloning.
5. Basic techniques of recombinant DNA technology.
5.1 Isolation of nucleic acids.
5.2 Quantification of nucleic acids: standard spectrophotometric assay and NanoDrop assay.
5.3 Agarose gel electrophoresis and sizing.
5.4 Transfer and immobilization of nucleic acids from the gel to a solid support (blotting).
5.5 Nucleic acid hybridization using labeled probes.
5.6 DNA sequencing.
5.7 Making LB agar plates and streaking of bacteria.
5.8 Inoculation of bacterial cultures and preparation of glycerol stocks for long term storage.
6. Introduction to expression vectors.

Bridging Courses

In order to understand the contents of the course, students should possess a good knowledge of basic Molecular biology.

Learning Achievements (Dublin Descriptors)

Students are expected to become familiar with the most popular techniques applied in gene cloning.

Teaching Material

The teaching material prepared by the lecturer in addition to recommended textbooks (such as for instance slides, lecture notes, exercises, bibliography) and communications from the lecturer specific to the course can be found inside the Moodle platform › blended.uniurb.it

Didactics, Attendance, Course Books and Assessment

Didactics

Learning activity will mainly consist of lab experiences. Lectures will be intended to supply the theoretical knowledge of the leading techniques employed in molecular biology.

Attendance

Students must attend at least 2/3 of lab hours to be admitted to oral examination.

Course books

• J. W. Dale, M. von Schantz, N. Plant. DAI GENI AI GENOMI, EdiSES, 2013.
• T. A. Brown. BIOTECNOLOGIE MOLECOLARI, Principi e tecniche, Zanichelli, 2007.
• S. Carson, H. Miller, D. S. Witherow. MOLECULAR BIOLOGY TECHNIQUES. A Classroom Laboratory Manual. Pub date: Dec 16, 2011. Elsevier Science & Technology.
• D. P. Clark, N. J. Pazdernik. BIOTECHNOLOGY, Academic Cell, Elsevier Inc, 2015.
• K. Wilson, J. Walker. BIOCHIMICA E BIOLOGIA MOLECOLARE, Principi e tecniche, Raffaello Cortina Editore, 2006.

Additional educational material or scientific reviews will be made available during the course.

Assessment

Oral examination.
Lab notebook evaluation.

Additional Information for Non-Attending Students

Didactics

Learning activity will mainly consist of lab experiences. Lectures will be intended to supply the theoretical knowledge of the leading techniques employed in molecular biology.

Attendance

Students must attend at least 2/3 of lab hours to be admitted to oral examination.

Course books

• J. W. Dale, M. von Schantz, N. Plant. DAI GENI AI GENOMI, EdiSES, 2013.
• T. A. Brown. BIOTECNOLOGIE MOLECOLARI, Principi e tecniche, Zanichelli, 2007.
• S. Carson, H. Miller, D. S. Witherow. MOLECULAR BIOLOGY TECHNIQUES. A Classroom Laboratory Manual. Pub date: Dec 16, 2011. Elsevier Science & Technology.
• D. P. Clark, N. J. Pazdernik. BIOTECHNOLOGY, Academic Cell, Elsevier Inc, 2015.
• K. Wilson, J. Walker. BIOCHIMICA E BIOLOGIA MOLECOLARE, Principi e tecniche, Raffaello Cortina Editore, 2006.

Additional educational material or scientific reviews will be made available during the course.

Assessment

Oral examination.
Lab notebook evaluation.

Notes

The student can request to sit the final exam in English with an alternative bibliography.

« back Last update: 08/02/16

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