Course detail

Laboratory Classes in Molecular Biotechnology

FCH-MC_MOB_PAcad. year: 2012/2013

Safety of work in microbiological and biotechnological laboratory. Arrangement of PCR laboratory. Cultivation of Lactobacillus gasseri cells for isolation of chromosomal DNA. Cell lysis. Removal of proteins using phenol extraction. Precipitation of DNA with ethanol. Verification of DNA integrity using agarose gel electrophoresis. Spectrophotometric estimation of concentration and purity verification of DNA. Preparation of PCR mixture for amplification of Lactobacillus gasseri using purified DNA as DNA matrix. Negative controls. PCR amplification. Detection of PCR products using agarose gel electrophoresis. Estimation of length of amplified fragment using external DNA standards. Sensitivity of PCR. Isolation of DNA from dairy product (yoghurt) using magnetic particles. Identification of Lactobacillus DNA in dairy product (yoghurt) by real time PCR. Testing of production of antimicrobial agents by probiotic strain Lactobacillus gasseri using cultivation methods and molecular diagnostic ones. Test.

Language of instruction

Czech

Number of ECTS credits

Mode of study

Not applicable.

Guarantor

doc. RNDr. Alena Španová, CSc.

Department

Institute of Food Science and Biotechnology (ÚCHPBT)

Learning outcomes of the course unit

Practical aspects of basic experimental techniques used in DNA analysis.Preparation of poster from own experimental results..

Prerequisites

It is necessary pass out lectures Bioanalytical methods.

Co-requisites

Not applicable.

Planned learning activities and teaching methods

Teaching methods depend on the type of course unit as specified in the article 7 of BUT Rules for Studies and Examinations.

Assesment methods and criteria linked to learning outcomes

There will be prepared 4 protocols based on own results in the form of a poster during the course. Laboratory course is finished by a test.

Course curriculum

1. Safety of work in microbiological and biotechnological laboratory. Arrangement of PCR laboratory. 2. Cultivation of probiotic Lactobacillus gasseri culture for isolation of bacterial DNA. Estimation of purity of bacterial culture. 3. Cell lysis and preparation of crude cell lysate. 4. Removing of proteins using phenol extraction. Precipitation of DNA with ethanol. 5. Determination of DNA integrity using agarose gel electrophoresis. Visualisation of DNA and documentation of the gel.
6. Spectrophotometric estimation of concentration and purity of DNA.DNA dilution. 7. Preparation of PCR mixtures for specific amplification of DNA Lactobacillus using purified DNA as DNA matrix. Estimation of PCR sensitivity. Negative controls. 8. PCR amplification. Detection of PCR products using agarose gel electrophoresis. 9. Estimation of length of amplicons using external DNA standards and program Bionumerics.
10. Isolation of DNA from dairy product (probiotic drink) using magnetic separation with magnetic particles P(HEMA-co-GMA). 11. Identification and quantification of Lactobacillus DNA in dairy product (probiotic drink ) by PCR v reálném čase.
12. Testing of antimicrobial compounds production. Amplification of gene coding bacteriocin in probiotic strain L. gasseri.
13. Test.

Work placements

Not applicable.

Aims

The laboratory course covers basic methodologies used in the work DNA and DNA amplification using polymerase chain reaction. The next aim is preparation of poster.

Specification of controlled education, way of implementation and compensation for absences

Attendance in laboratory course is obligatory.

Recommended optional programme components

Not applicable.

Prerequisites and corequisites

Not applicable.

Basic literature

Španová A., Rittich B.: Analýza vybraných druhů baktérií mléčného kvašení pomocí metod molekulární biologie (CS)

Type of course unit

Guided consultation in combined form of studies

13 hod., optionally

Teacher / Lecturer

doc. RNDr. Alena Španová, CSc.

Laboratory exercise

26 hod., compulsory

Teacher / Lecturer

doc. RNDr. Alena Španová, CSc.
Ing. Barbora Gregušová
Ing. Štěpánka Trachtová, Ph.D.
Mgr. Kristýna Turková, Ph.D.