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Last update: Mgr. Roman Leontovyč, Ph.D. (18.03.2019)
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Last update: Mgr. Roman Leontovyč, Ph.D. (18.03.2019)
Mandatory: Printed tutorials for practical tasks, with a brief overview of the methods. Presentations
Recommended: D. Thienpont, F. Rochette, O. F. J. Vanparijs. Diagnosing helminthiasis by coprological examination. 2nd ed. Beerse, Belgium: Janssen Research Foundation 1986. ISBN BWB30960491
Technical guide for ELISA available at https://www.seracare.com/globalassets/seracare-resources/tg-protocols-and-troubleshooting.pdf
T.A. Brown. Essential Molecular BiologyVolume 1. 2nd ed. Oxford, UK: Oxford University Press, 2000. ISBN 0-19-963643
John M. S. Bartlett, David Stirling. Methods in Molecular Biology, vol. 226 PCR Protocols.2nd ed. Totowa, New Jersey: Humana Press, 2003. ISBN 0-89603-642-1
Brenda D. Spangler. Methods in molecular biology and protein chemistry – Cloning and characterization of an enterotoxin subunit. West Sussex, UK: John Wiley & Sons, Ltd., 2002. ISBN 0-470-84360-8
Daniel M. Bollag, Stuart J. Edelstein. Protein methods New York, NY: Wiley-Liss, 1991. ISBN 0-471-56871-6 |
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Last update: Mgr. Roman Leontovyč, Ph.D. (18.03.2019)
Active participation in the course is mandatory (one excused absence is allowed). The short test will be written before the selected lessons focused on the topic of the lesson. The results of the continuous tests will be taken into account in the final test. All tests will be in the written form where “multiple-answer” testing (none to all answers correct) will be applied, followed by short-answer questions. Ambiguous results can lead to the oral part of the exam. |
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Last update: Mgr. Roman Leontovyč, Ph.D. (18.03.2019)
1. Fundamentals of the laboratory work
Theoretical part: - safety, weights & measures, the fundamentals of pipette handling, introduction of the basic laboratory equipment (centrifuges – balance, RPM/g, flowbox.), sterile work. Practical part: - calculations: unit conversions, volume concentration, molar concentration - preparation of the solutions – dilution, pH adjustment and measurement - preparation of the growth media and agar plates
2. Dry smear preparation and staining, handling the laboratory mouse Theoretical part: - parasitic protozoa transmitted by insects; laboratory animals in parasitology and microbiology Practical part: - blood smear infections of Trypanosoma brucei, Plasmodium berghei - thick drop – diagnostics of Plasmodium berghei - dry smears - Giemsa-Romanowsky staining, Dade Diff-Quik staining - handling of the laboratory mouse, IP inoculation
3. Cloning, quantitative experiments with protozoa Theoretical part: - growth of the microbial cultures, growth curve, calculation of the generation time; cloning methods, colony-forming efficiency Practical part: - usage of the Burker counting chamber - cell counting - handling the liquid media, maintaining cells in culture - cloning by limiting dilution (Trichomonas vaginalis)
4. Cultivation techniques Theoretical part: - types of microbial cultures, cultivation media, procedures in cryopreservation of the protozoa Practical part: - evaluation of the cloning experiment - axenization of the protozoan cultures, making of the migration tube - cryopreservation techniques, cryopreservation of the protozoa, cryobank demonstration
5. Coprological methods Theoretical part: - principles of material collection, hygienic measures, requirements, ocular micrometre calibration, methods overview Practical part: - coprological examination using flotation, sedimentation, larvoscopy
6. ELISA Theoretical part: - principle, usage in diagnostics Practical part: - antibody detection against the Toxoplasma gondii
7. Nucleic acids I Theoretical part: - DNA structure (base pairing), principle of DNA isolation on silicate, principle of isolation of DNA/RNA using TRIzol Reagent, RNA integrity control Practical part: - DNA isolation – silicate columns - DNA precipitation - determination of DNA concentration
8. Nucleic acids II Theoretical part: - PCR principle, PCR modulations (nested PCR, RT-PCR, q-PCR, multiplex PCR) Practical part: - PCR reaction preparation, operating the thermocycler - agarose gel electrophoresis - DNA purification from an agarose gel
9. Rcombinant proteins I Theoretical part: - principle of the expression of foreign protein in bacterial cell, principle of the protein electrophoresis SDS PAGE Practical part: - cryopreservation of the bacterial cultures - assessment of the protein profile of the bacterial colony using of SDS PAGE (protein solubility test)
10. Rcombinant proteins II Theoretical part: - isolation of the recombinant protein in native and denaturing conditions, principle of the isolation of the polyhistidine tagged protein Practical part: - isolation of the recombinant protein using affinity chromatography (His-Tag)
11. Mass spectrometry, immunoblot Theoretical part: - mass spectrometry analysis of biological samples (diagnostics, recombinant proteins identification, proteome analysis), sample preparation Practical part: - immunoblot – identification of His-tagged protein |