Katedra molekulární biologie a genetiky
Department of molecular biology and genetics

Advanced methods of molecular biology

Please read the Sign-up guide section carefully before signing up for a course.

Sign-up Guide

Before signing up for a course please take a look at "The current state of free spots" to verify that the course you chose has a spot for you (link below). Each course has an individual minimum and a maximum number of attendees. The minimal number of attendees to open the course is listed in the course label and is also indicated by green colour in the course column. After you pick a course with free spot, click on the "Course Signup" button that will redirect you to a form, where you fill out your name and email address. You can verify that you have been signed up by checking "The current state of free spots" again. Your name should appear in an appropriate column within a few minutes of signing up.

Please note that if your name appears in the red cell it means that you exceed the capacity of a course and you need to sign up to a different course.

The current state of free spots

Course Descriptions

Course 1
Guarantor: Adam Bajgar
Name of the technique: qPCR expression analysis
Responsible person name: Gabriela Krejčová
Responsible person email: krejcovagabriela@seznam.cz
Duration of the course: 12 hours
Short description: Practice will consist of a selection of concrete gene, work with analysis and design of
qPCR primers. The tissue samples will be used for RNA isolation and reverse transcription. qPCR by using
SYBR green will be followed by data analysis by method double delta.

Course 1 Sign Up

Course 2
Guarantor: Tomáš Doležal
Name of the technique: Advanced Drosophila genetics – generation and analysis of mitotic clones
Responsible person name: Tomáš Doležal
Responsible person email: tomas.dolezal@prf.jcu.cz
Duration of the course: 3 days
Short description: Theoretical learning of advanced Drosophila genetics and crosses for mitotic clone
generation, fly handling and phenotyping under the microscope, larval dissection and microscopy
sample preparation, confocal microscopy analysis of clones.

Course 2 Sign Up

Course 3
Guarantor: David Doležel
Name of the technique: Systemic RNA interference in insects
Responsible person name: David Doležel
Responsible person email: david.dolezel@entu.cas.cz
Duration of the course: 4-5 afternoons
Short description:
Day 1: PCR to prepare a template for RNA transcription (theory during the PCR reaction),
Day 2: Product purification + in vitro transcription (O/N)
Day 3: dsRNA annealing & Injection into P. apterus
Day 4: Phenotype assessment – 2 weeks later – 2hrs during the afternoon

Course 3 Sign Up

Course 4
Guarantor: Alexander W. Bruce
Name of the technique: Microinjections and immuno-fluorescence staining of mouse embryos
Responsible person name: Pablo Bora (Ph.D. student)
Responsible person email: borapa00@prf.jcu.cz
Duration of the course: 4-5 days
Short description: Microinjections (of mRNAs, short RNAs or proteins) are a common technique used for
the gene down-regulation or overexpression in mammalian oocytes and embryos. We will inject 1 cell of
mouse 2-cell stage embryos with fluorescently-tagged mRNA, embryos will be cultured until the early
blastocyst stage and then they will be fixed and immuno-stained for a marker protein of trophoblast cell
lineage (future placenta). Confocal microscopy will be used to visualise injected fluorescent protein and
immuno-stained trophoblast cell lineage marker, differentiating between the progeny of injected and
the non-injected cell at the 2-cell stage, as well as between the cells that will contribute to the placenta
(presence of trophoblast marker protein) and the embryo itself (absence of trophoblast marker protein).

 Detailed protocol Course 4 Sign Up

Course 5
Guarantor: Petr Nguyen
Name of the technique: Fluorescence in situ hybridization
Responsible person name: Petr Nguyen
Responsible person email: petr.nguyen@prf.jcu.cz
Duration of the course: 3 days
Short description: Students will learn how to make chromosome preparations, construct fluorescently
labelled probes and hybridize them to chromosomes. Probes can be constructed from a whole genome,
clone from the genomic library, repetitive sequence or single-copy gene.

 Detailed protocol Course 5 Sign Up

Course 6
Guarantor: Tomáš Korytář
Name of the technique: Flow cytometry and cell sorting
Responsible person name: Tomáš Korytář
Responsible person email: tkorytar@paru.cas.cz
Duration of the course: 3 days
Short description: In this course, students will learn the basic principles of flow cytometry and its
applications in life sciences on routinely used assays. The key aim is to provide students with the
knowledge necessary for performing any flow cytometry application from the design of the experiment,
design of antibody panels, sample acquisition and subsequent analysis and data presentation.

 Detailed protocol Course 6 Sign Up

Course 7
Guarantor: Zdeněk Paris
Name of the technique: Denaturing polyacrylamide gel electrophoresis and radioactive detection of
small non-coding RNAs by Northern blot
Responsible person name: Zdeněk Paris
Responsible person email: parda@paru.cas.cz
Duration of the course: 4 days
Short description:Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures
secondary RNA structures and is used for their separation in a polyacrylamide gel matrix based on the
molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single
nucleotide, can be separated using this method. The migration of the sample is dependent on the
chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular
weight fragments. The combination of urea and temperatures of 45-55 °C during the gel run allows for
the separation of unstructured RNA molecules. After the PAGE, the gel is transferred to a nitrocellulose
membrane and hybridized with a P32 radiolabeled oligonucleotide probe to visualize the specific RNA
species using the Phosphoimager.

Course 7 Sign Up

Course 8
Guarantor: Roman Sobotka
Name of the technique: 2D native/SDS electrophoresis combined with immunodetection
Responsible person name: Petra Skotnicová
Responsible person email: skotnicova@alga.cz
Duration of the course: 2.5 days
Short description: During the course, students will learn to set a large electrophoretic system (Bio-Rad
Protean xi cell), to pour and run blue-native and clear-native 8-14% gradient gels with samples of
membrane protein complexes. Gel strips from the first dimension will be separated overnight on a 12-
20% SDS gel, stained with SYPRO Orange, blotted onto PVDF membrane and probed with selected

Course 8 Sign Up

Course 9
Guarantor: Roman Sobotka
Name of the technique: Expression and purification of His-tagged proteins
Responsible person name: Petra Skotnicová
Responsible person email: skotnicova@alga.cz
Duration of the course: 2 days
Short description: Students will learn the transformation of E. coli by electroporation, cultivation of E. coli
cells, preparation cell lysate by sonication, purification of His-tagged fluorescent proteins on a nickel
column, analysis of purified proteins on a small SDS gel, Coomassie stain.

Course 9 Sign Up

Course 10
Guarantor: Hana Sehadová
Name of the technique: Classical histological techniques and light microscopy
Responsible person name: Hana Sehadová, Markéta Vrchotová
Responsible person email: sehadova@yahoo.com, ratuska@centrum.cz
Duration of the course: 2 days
Short description: 1. Preparation of paraffin sections of insect tissues and blood smear, 2. Mallory and
Giemsa staining, 3. Observation of obtained samples in the light microscope.

Course 10 Sign Up

Course 11
Guarantor: Alena Zíková
Name of the technique: Detection of high molecular weight respiratory complexes using native PAGE
and subsequent Wester blot analysis
Responsible person name: Míša Kunzová (kunzova.michaela@gmail.com)/ Carolina Hierro Yap
Responsible person email: kunzova.michaela@gmail.com, carol.hierro@yahoo.es
Duration of the course: 2.5 days (beginning  of May)
Short description: Students will get an introduction into different types of native gel electrophoresis that are used to analyze
high molecular weight complexes. They will get to understand the difference between SDS and native
PAGE, the principle of native PAGE, and its applications. During the practical session, they will work with
purified mitochondria from Trypanosoma brucei and they will use different detergents to obtain
mitochondrial lysates. The cleared lysates will be separated on Blue and High-Resolution Clear Native
(BNE a hrCNE) gels. One group of gels will be stained with coomassies to detect all respiratory chain
complexes and the second group of gels will be transferred onto the PVDF membrane followed by Western
blot analysis. The students will use antibodies against complexes I, II, III, IV and V to detect these
assemblies as well as supercomplexes.

 Detailed protocol Course 11 Sign Up

Course 12
Eva Doleželová
Name of the technique: Measurement of mitochondrial respiration by High-Resolution Respirometry
(HRR) using the O2k-FluoRespirometer
Responsible person name: Carolina Hierro Yap
Responsible person email: carol.hierro@yahoo.es
Duration of the course: 1.5 days (beginning  of May)
Short description: The students will get a theoretical introduction into the High-Resolution Respirometry, the principle and
what it is used for. Then during the practical session, they will measure respiration in intact and digitonin
permeabilized cells adding different substrates and inhibitors. From these experiments, the students will
be able to determine basal and stimulated respiration, define state 3 and 4 respiration as well maximal
capacity of the mitochondrial electron transport chain.

 Detailed protocol Course 12 Sign Up

Course 13
Guarantor: Marek Jindra
Name of the technique: Western blot
Responsible person name: Marek Jindra
Responsible person email: jindra@entu.cas.cz
Duration of the course: 2 days
Short description: In this hands-on course students will analyze the expression of specific proteins of
interest either in insect tissues or in cultured cells. They will prepare tissue/cell extracts, prepare and
run denaturing polyacrylamide gel electrophoresis, and detect proteins after immunoblotting using
antibodies and chemiluminescence.

Course 13 Sign Up

Course 14
Guarantor: Petr Nguyen
Name of the technique: NGS data processing
Responsible person name: Petr Nguyen
Responsible person email: petr.nguyen@prf.jcu.cz
Duration of the course: 3 days
Short description: Students will learn basic processing of NGS data
Detailed protocol: Bash basics; cluster computing within the Metacentrum infrastructure; data download; quality check
and filtering; de novo assembly and read mapping.

Course 14 Sign Up

Course 15
Guarantor: Lenka Gahurová, Iva Mozgová
Name of the technique: RNA-seq library preparation and data analysis
Responsible person name: Iva Mozgová, Lenka Gahurová
Responsible person email: mozgova@umbr.cas.cz, lveselovska@prf.jcu.cz
Duration of the course: 2 days
Short description: RNA sequencing is used to study changes in RNA expression at the genome-wide level
either at different time point or at different conditions. This course will teach you: 1. How to find the
best approach and the best kit for RNA-seq library preparation of your samples 2. How to find the best
sequencing strategy 3. How to check the quality of your library 4. How to check the quality of your data
5. How to analyse your data to identify differentially expressed genes.

 Detailed protocol Course 15 Sign Up

Course 16
Guarantor: Michal Žurovec
Name of the technique: Transfection of insect cell lines
Responsible person name: Václav Brož
Responsible person email: vasa.broz@centrum.cz
Duration of the course: 3 days
Short description: We will show students different types of insect cell cultures, teach them DNA
transfection to the cells and detection of marker expression.

 Detailed protocol Course 16 Sign Up


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