Bi6405 Methods of Molecular Biology - practice

Faculty of Science
Spring 2014
Extent and Intensity
0/3/0. 3 credit(s). Type of Completion: z (credit).
Teacher(s)
doc. Mgr. Petr Beneš, Ph.D. (seminar tutor)
doc. RNDr. Jakub Neradil, Ph.D. (seminar tutor)
Mgr. Lucia Knopfová, Ph.D. (seminar tutor)
prof. RNDr. Jan Šmarda, CSc. (seminar tutor)
Guaranteed by
prof. RNDr. Jan Šmarda, CSc.
Department of Experimental Biology – Biology Section – Faculty of Science
Contact Person: prof. RNDr. Jan Šmarda, CSc.
Supplier department: Department of Experimental Biology – Biology Section – Faculty of Science
Timetable
Thu 7:00–12:50 D36/216, Fri 9:00–14:50 D36/216
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 practical skills in microbiology, biochemistry and molecular biology as, for example, preparation of buffers, cultivation media, sterile liquide handling, inoculation, 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
Course objectives
Main objectives can be summarized as follows: to learn the basic techniques of DNA manipulation in vitro to obtain recombinant DNA molecules to learn the ways of successful purification of plasmid DNA from bacterial cells; to cut the purified plasmid DNA using various restriction enzymes; to become familiar with approaches to modify recessed termini of DNA to seal DNA molecules using DNA ligases; to perform separation of DNA and protein molecules by gel electrophoresis; to elute DNA molecules out of a agarose gels, to perform immunoblotting and to learn ways of successful transfection of plasmid DNA into eukaryotic cells. At the end of the course students should be able to perform these experiments independently and to applied in correct way to address specific biological phenomena.
Syllabus
  • 1. Inoculation and cultivation of bacterial cells having appropriate plasmids. 2. Isolation of plasmid DNA from bacterial cells using Qiagen columns. 3. Restriction analysis of plasmid DNA and modification of 5´ overhang termini using DNA polymerase. 4. Agarose electrophoresis of fragmented DNA. 5. Elution of appropriate fragments from a gel. 6. Ligation of appropriate DNA molecules. 7. Transformation of competent bacterial cells with ligation mixture. 8. Selection of transformants. 9. Screening of transformants for the presence of recombinant plasmid (plasmid minipreparation, restriction analysis, electrophoresis). 10. Recombinant plasmid transfection into mammlian cells. 11. Cell lysis, protein electrophoresis and immunoblotting.
Literature
  • 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
Brief lecture follwed by practical laboratory training, class discussion.
Assessment methods
Credits are given for active work at a bench, for participation in each lesson and for elaborating detailed protocols of each step of the procedure used, including results and conclusions obtained.
Language of instruction
Czech
Follow-Up Courses
Further Comments
Study Materials
The course is taught annually.
Listed among pre-requisites of other courses
The course is also listed under the following terms Spring 2008 - for the purpose of the accreditation, Spring 2011 - only for the accreditation, Spring 2003, Spring 2004, Spring 2005, Spring 2006, Spring 2007, Spring 2008, Spring 2009, Spring 2010, Spring 2011, Spring 2012, spring 2012 - acreditation, Spring 2013, Spring 2015, Spring 2016, Spring 2017, spring 2018, Spring 2019.
  • Enrolment Statistics (Spring 2014, recent)
  • Permalink: https://is.muni.cz/course/sci/spring2014/Bi6405