Lahore University of Management Sciences

BIO 300 Methods on Molecular Biology Fall 2013 Instructor Dr. Muhammad Tariq, Dr. Saima Anwar, Dr. Zakir Ullah & Dr. Aziz Mithani Room No. 9-415 A , 9-420A , 9-413 A , 9-419 A Office Hours N/A Email m.tariq@lums.edu.pk, Saima.anwar@lums.edu.pk ; zakirullah@lums.edu.pk; aziz.mithani@lums.edu.pk Telephone 8218, 8352, 8217, 8397 Secretary/TA N/A TA Office Hours N/A Course URL (if any) N/A Course Basics Credit Hours 4 Lecture(s) Nbr of Lec(s) Per Week N/A Duration N/A Recitation (per week) Nbr of Rec (s) Per

Week N/A Duration N/A

Lab (if any ) per week Nbr of Session(s) Per Week

3 Duration 4 hours/day

Tutorial (per week) Nbr of Tut(s) Per Week

N/A Duration N/A

Course Distribution Core Yes Elective No Open for Student Category Biology Majors Closed for Student Category - COURSE DESCRIPTION This lab based course introduces modern tools of molecular cloning; protein expression and purification which are used to understand cell and molecular biology of cellular function. In addition, students are introduced to experimental model system Drosophila melanogatser which is used to perform classical genetics by crossing various genetic mutants as well as different tissues of fruit fly larvae are dissected to understand development, inducible gene expression, active and silent genes. Different modules of the lab course are designed so that students address a specific question by utilizing different recombinant DNA technology tools. Students will design and carry out experiments to address specific questions about genes involved in the regulation of transcriptional regulation, cellular development, cell fate determination and cell death etc. COURSE PREREQUISITE(S)

BIO100, BIO101 BIO216, BIO221, BIO212 Instructors consent

COURSE OBJECTIVES

Introduce students to modern concepts and techniques in molecular cloning and protein purification Introduce students to form a scientific question and address it by different tools possible Introduce genetics, molecular, biochemical and cytological approaches to address same scientific question

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Learning Outcomes

Students must be able to: Develop a scientific approach to address a biological question about cellular function Perform experiments independently by having a specific protocol and must also be able to search for experimental protocols Address a cellular function through multiple approaches like genetic, molecular and biochemical.

Grading Breakup and Policy There will be no Midterm and Final Exam. The whole grading will be based on lab and will be decided by individual instructor for his/her modules Assignment(s): Home Work: Quiz(s): Class Participation: Attendance: Midterm Examination: Project: Final Examination: Examination Detail

Midterm Exam

Yes/No: NO Combine Separate: Duration: Preferred Date: Exam Specifications:

Final Exam

Yes/No: NO Combine Separate: Duration: Exam Specifications:

COURSE OVERVIEW

Week/ Lecture/ Module

Topics Recommended Readings Objectives/ Application

Module 1

1. Drosophila as a Model for Genetics and Development

Drosophila Embryonic Staging. Dissection of IIIrd Instar Larvae for isolation

of Imaginal Discs. Different stages of Drosophila development. Balancer Chromosomes and markers 2. Inheritance pattern of Eye Color in

Drosophila

3. Chromosome Mapping of a Transgene (Hsp83EGFP)

Selected readings for each module from books mentioned in Textbook(s)/Supplementary Readings

Students will be introduced to basic genetic tools which can be used to answer different questions by using Drosophila melanogaster as a model organism

Module 2

1. Polytene Chromosomes: Identification of heat shocked Puffs and Banding Patterns

2. Developmental Tissue Specific Gene

Expression

To study the temporal and tissue specific gene expression in vivo. Students will also learn what are

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3. Identification of Heterochromatin and

Euchromatin regions on Polytene Chromosomes

epigenetic marks which can be used to differentiate between euchromatin and heterochromatin regions of chromosomes.

Module 3 Induction of Somatic Clones using Flp-FRT system: Effect of Homozygous Mutations of Polycomb on Expression of Homeotic Genes (Ubx).

Students will learn how they can study the phenotype of recessive lethal mutations

Module 4 Chromatin Immunoprecipetation (ChIP).

Students will learn a technique which can be used to study the binding profiles of transcription factors or any other DNA binding proteins

Module 5

Reverse Genetics: To see the effect of knockdown of Polycomb and Trithorax group proteins on their target genes in cell culture

They will learn how dsRNA can be used to silent the target genes at post-transcriptional level (PTGS) in Drosophila cell culture.

Module 6

Introduction Preparation of chemically competent E. coli

cells Bacterial Transformation with plasmid DNA Plasmid purification Gel electrophoresis agarose Restriction analysis and plasmid mapping

Basic Molecular biology tools kit: They will learn how they can clone and express their gene of interest.

Module 7

Plasmid preparation (restriction and dephosphorylation)

Purification of vector DNA from agarose gel Ligation Transformation and plating Identification of recombinant clones

(inoculate O/N cultures) DNA isolation from buccal swabs and

chicken, cow and sheep liver Amplification of mitochondrial DNA cox-2

gene segment via PCR Identification of recombinant plasmids Mini-preparation of plasmid DNA Digestion with restriction enzyme

Basic Molecular biology tools kit:

Module 8

Purification of PCR products for DNA sequencing

Multiplex PCR of dystrophin gene Analysis of multiplex PCR products on

polyacrylamide gels Transformation of E. coli for protein

expression Isolation of total RNA from HeLa cells RT-PCR Grow E. coli for protein expression (collect

cells)

Module 9

Affinity purification of recombinant protein Separate fractions on SDS-PAGE Coomassie stain half; electroblotting of the

other Western blotting Expression and purification of E. coli RNA

polymerase

Students will learn basic protein biochemistry

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Module 10 Isolation and characterization of satellite DNA from bovine genomic DNA

Textbook(s)/Supplementary Readings

A Handbook of Drosophila Development (1982) Robert Ransom (ed.). Elsevier Biomedical Ashbumer, M. (1989) Drosophila: A Laboratory Handbook and Drosophila: A Laboratory Manual Cold Spring Harbor Laboratory Press Drosophila. A Practical Approach (1998) Roberts, D.B. (ed.). IRL Press Lawrence, P. A. (1992)The Making of a Fly: The Genetics of Animal Design Blackwell Scientific Publications Rubin, G. M. (1988) Drosophila melanogaster as a ExperimentalOrganism. Science 240, 1453-1459 Ralph J. Greenspan (1997) Fly Pushing CSHL Press Campos-Ortega, J. A. and Hartenstein, V. (1997) The Embryonic Development of Drosophila melanogaster. Springer-Verlag (Berlin, Heidelberg).

Week/ Lecture/ ModuleObjectives/RecommendedTopicsApplicationReadings