Bi6400c Methods of Molecular Biology - practice

Faculty of Science
Spring 2023
Extent and Intensity
0/2/0. 2 credit(s). Type of Completion: z (credit).
Teacher(s)
doc. Mgr. Petr Beneš, Ph.D. (seminar tutor)
Mgr. Jarmila Navrátilová, Ph.D. (seminar tutor)
Mgr. Lucia Knopfová, Ph.D. (seminar tutor)
Mgr. Jiří Kohoutek, Ph.D. (seminar tutor)
Guaranteed by
doc. Mgr. Petr Beneš, Ph.D.
Department of Experimental Biology - Biology Section - Faculty of Science
Contact Person: doc. Mgr. Petr Beneš, Ph.D.
Supplier department: Department of Experimental Biology - Biology Section - Faculty of Science
Prerequisites
( Ex_3065 Molekulární biologie || Imp_9115 Molekulární biologie || B3120 Molecular and cell biology || B4030 Molecular biology || B5740 Molecular biology || B6130 Molecular biology || B7940 Molecular biology || B4020 Molecular biology || Bi4020 Molecular biology || Bi4035 Molecular biology - lab.pract. ) && NOW ( Bi6400 Methods of molecular biology ) && ! B6405 Molecular Methods - practice
The course requires basic computer competency, fundamental knowledge of molecular biology and bioinformatics and practical skills in microbiology, biochemistry and molecular biology as, for example, manipulation with automatic pipettes, preparation of buffers and cultivation media, sterile liquide and bacterial cultures handling, centrifugation, and so on.
Course Enrolment Limitations
The course is only offered to the students of the study fields the course is directly associated with.
fields of study / plans the course is directly associated with
there are 13 fields of study the course is directly associated with, display
Course objectives
The aim of this course is to prepare and realize a project for silencing target gene expression using CRISPR/Cas9 a shRNA methodology.
Learning outcomes
At the end of this course, students will be able to to identify CRISPR/Cas9 and siRNA targets in sequence of gene of interest using public bioinformatic tools, to create and purify recombinant CRISPR/Cas9 and shRNA vectors, to transfect these vectors into mammalian cells, to analyze mutational status of target gene and to evaluate gene expression. Students will be ale to use the acquired knowledge to prepare and to implement a project analyzing the function of selected genes in diverse biological processes.
Syllabus
  • 1. Introductory lecture – gene edditing, CRISPR/Cas9, RNA interference, bioinformatic tools – gene sequence search, sequence alignment, design of CRISPR/Cas9 and shRNA targets, open reading frame finder. 2. Preparation of individual written protocols by students with the aim to design CRISPR/Cas9 and shRNA vectors for target gene. 3. Evaluation of protocols by a teacher. 4. Implementation of experimental protocol in the lab – preparation and purification of recombinant CRISPR/Cas9 and shRNA vectors, mammalian cells transfection, cells harvestion and lysis, determination of protein concentration, SDS-PAGE and immunoblotting, genomic DNA isolation, polymerase chain reaction, DNA sequencing. 5. Analysis of mutational status of gene of interest, finnalization of individual protocols.
Literature
  • YAMAMOTO, T. Targeted Genome Editing Using Site-Specific Nucleases, ZFNs, TALENs, and the CRISPR/Cas9 system. 2015. Springer. 205 pp. ISBN 978-4-431-55226-0.
  • MOULDY, S. RNA Interference Challenges and Therapeutic Opportunities. 2015. New York : Humana Press : Springer. ISBN: 978-1-4939-1537-8.
  • GREEN, Michael R. and Joseph SAMBROOK. Molecular cloning : a laboratory manual. 4th ed. New York [N.Y.]: Cold Spring Harbor Laboratory Press, 2012. xxxiii, s. ISBN 9781936113422. info
Teaching methods
Introductory lecture, individual written project prepared by students followed by practical laboratory training, class discussion.
Assessment methods
Credits given for project preparation, participation in practical lessons and experimental data processing and analysis.
Language of instruction
Czech
Follow-Up Courses
Further Comments
The course is taught annually.
The course is taught: every week.
Listed among pre-requisites of other courses
The course is also listed under the following terms Spring 2020, Spring 2021, Spring 2022.
  • Enrolment Statistics (Spring 2023, recent)
  • Permalink: https://is.muni.cz/course/sci/spring2023/Bi6400c