CAF
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Training Initiative: The Workshops

  1. Mass Spectrometry of Small Molecules: LC-MS
  2. Mass Spectrometry of Small Molecules: GC-MS
  3. Mass Spectrometry Based Proteomics (Tygerberg Campus)
  4. Solution State NMR
  5. Flow Cytometry and Cell Sorting
  6. Super-resolution Microscopy
  7. STEM: Sample Preparation and Imaging
  8. Correlative Light and Electron Microscopy
  9. Introduction to X-ray Micro- and Nano-CT
  10. Nucleic Acid Preparation, QC and Library building for Next Generation Sequencing
  11. 3D Neuro-biomechanical Analysis (Tygerberg Campus)

There will be three Mass Spectrometry training workshops, two for non-proteomic applications and one for proteomic applications. Other units presenting more than one workshop include: the Fluorescent Microscopy Unit (Flow Cytometry and Super-resolution Microscopy) and the SEM Unit (STEM and Correlative Light and Electron Microscopy). All workshops are described below.

Mass Spectrometry (MS)

1. Mass Spectrometry of Small Molecules: LC-MS

Presenters: Dr Marietjie Stander, Ms Meryl Patience and Mr Malcolm Taylor

Duration: 1 Day

2. Mass Spectrometry of Small Molecules: GC-MS

Presenter: Mr Lucky Mokwena

Duration: 1 Day

The training on small molecules (and to some extent polymers) will be separated into an LC-MS and GC-MS component. Each component will be presented as a full day workshop and will be repeated twice during the training week. Both workshops will have a theoretical and practical component and will incorporate the topics outlined below.

Topics that will be covered include:

  • Overview of the advantages and disadvantages of the technique
  • Applications
  • Sample preparation
  • Different variations of the technique
  • Hardware
  • Software
  • Data processing and manipulation

3. Mass Spectrometry Based Proteomics (Tygerberg Campus)

Presenter: Dr Maré Vlok

Duration: 3 Days

After this 3-day workshop the student will know the basic principles of mass spectrometry, be able to prepare biological samples for mass spectrometry-based analysis and be able to use global databases for mass spectrometry-based data in order to identify proteins. Finally, the student will be able to recognise the value of mass spectrometry for their own research.

Course will comprise of the following:
  • Theoretical component: basic principles of mass spectrometry and proteomics, overview of various proteomics and mass spectrometry based applications.
  • Tutorials: Design of proteomics and mass spectrometry based experiments.
  • Practicals: Preparation of biological samples for mass spectrometry with the specific aim of proteomics based applications. Therefore, samples will be used from gels and will be cleaned, trypsin digested and finally prepared for identification by mass spectrometry. Data obtained from the practical will then be used for database searches in order to teach the student the basic principles that are important for database searches.
  • Discussions: After each component time is allocated for questions and answers, the course is also ended with an in depth discussion from the students and how they would apply mass spectrometry within their own projects.

Day 1:
Lecture: Introduction to Proteomics, sample extraction and preparation for digest
Practical: Prepare samples for in-gel and in-solution digest
Discussion: Any questions on the day?

Day 2:
Practical: Extraction and clean-up of peptides
Practical: Preparation of MS system
Lecture: Chromatographic separation and enrichment techniques
Lecture: Mass spectrometry
Lecture: Quantitative proteomics
Discussion: Questions?

Day 3:
Practical: Preparing database search
Practical: Investigating data
Lecture: Data analysis and statistics
Discussion: Students feedback on how they can apply Mass spectrometry based proteomics in their projects.

Nuclear Magnetic Resonance (NMR)

4. Solution State NMR

Presenter: Dr Jaco Brand

Duration: 1 Day

The purpose of the workshop is to connect the theory to applications in modern solution state NMR. The aim is to reintroduce participants to the theory and on the practical aspects of the technique. Examples from different application areas such as chemistry, biochemistry, pharmaceuticals, polymers and petrochemicals will be used to highlight the importance of the technique in structure elucidation, absolute stereochemistry determination as well as chemical composition determination and reaction kinetics. Structure determination is the main strength of the technique and will receive the most attention.

The following topics will be discussed:

Morning Session:
  • NMR theory lecture
  • Hands-on sample preparation techniques and choosing solvents for your samples
  • Running selected 1D and 2D experiments by participants on the instrument
  • Evaluating and processing data, Spinworks or Mestrenova processing
Afternoon Session:
  • What can I do with NMR?
  • From structure determination to reaction kinetics, metabolomics, medical and biological applications
  • Hands-on interactive session on the interpretation of 1D and 2D data of selected organic structures
  • Lecture on the use of Chiral shift reagents to determine absolute stereochemistry

Fluorescence Microscopy

5. Flow Cytometry and Cell Sorting

Presenter: Ms Rozanne Adams

Duration: 2 Days

This course introduces the participant to flow cytometry and the applications of this technology, with a special focus on sorting. Participants will get the chance for hands-on acquisition of samples to familiarise themselves with the work flow on a flow cytometer, the setup and calibration of the instrument and data analysis. Emphasis will be placed on sorting and students will get the chance to run a short sorting experiment themselves.

The content that will be covered includes:

  • Basic understanding of the components of a flow cytometer
  • Knowledge of key concepts: light and colour, scatter, laser delay, pulse area and width, spillover
  • Hands-on acquisition
  • Data analysis using FACSDiva and FlowJo
  • Setup and running of a sorting experiment

6. Super-resolution Microscopy

Presenters: Dr Ben Loos and Ms Lize Engelbrecht

Duration: 1 Day

The course introduces theory and applications for the subdiffraction limit microscopy techniques structured illumination (SR-SIM), photoactivation localization (PALM) and stochastic optical reconstruction (STORM) microscopy.

Morning session:
Theory session on super-resolution techniques
Discussion on sample preparation protocols
Calibration of the system for super-resolution microscopy

Afternoon session:
Calibration of the system for super-resolution microscopy
Hands-on acquisition of samples in the different super-resolution modules
Data processing of acquired images

Scanning Electron Microscopy

7. STEM: Sample Preparation and Imaging

Presenters: Ms Dumisile Lumkwana and Ms Madelaine Frazenburg

Duration: 2 Days

Day 1: Sample preparation for STEM

The first day of the workshop will be hosted by the CAF Fluorescent Microscopy Unit. This part introduces the participant to preparation of biological specimens for viewing with scanning transmission electron microscopy (STEM). Participants will be introduced to all the steps involved in sample preparation such as fixation, dehydration, embedding, polymerization, sectioning and staining. Participants will observe and might get a chance of sectioning semi-thick and ultra-thin slices with ultra-microtome.

Day 2: STEM imaging

The next day will be spent in the Scanning Electron Microscopy Unit where users will receive instruction on the use of the Scanning Transmission Electron Microscopy installed on the Zeiss Merlin FE SEM. Background theory on the system will be presented with some hands-on acquisition on the system to follow.

8. Correlative Light and Electron Microscopy

Presenters: Ms Lize Engelbrecht and Ms Madelaine Frazenburg

Duration: 1 Day

Morning session:
A short presentation will be followed by image acquisition on the confocal microscope in the Fluorescent Microscopy Unit. Various strategies to set up a CLEM experiment will be introduced. A selection of fluorescently labelled samples will be imaged during this session.

Afternoon session:

In the afternoon, the samples imaged in the morning on the confocal microscope will be transferred to the Scanning Electron Microscopy Unit and using the Shuttle and Find feature on the Merlin FE SEM, the same samples will be imaged and overlaid to produce correlative fluorescence and electron micrographs.

CT Scanner

9. Introduction to X-ray Micro- and Nano-CT

Presenter: Mr Stephan le Roux

Duration: 1 Day

The workshop will cover the basics of X-ray Micro-CT and Nano-CT.

Course content will include:

  • Specific examples
  • How the method works
  • What kind of information can be found
  • Advantages of X-ray 3D imaging

Systems will be demonstrated and prepared data will be used for demonstrating 3D data analysis. This will cover basic 3D visualization, image segmentation, defect analysis, part to part comparison and many more. The course focuses on new users and is ideal for researchers and students new to the field of non-destructive "X-ray microscopy".

DNA Sequencer

10. Nucleic Acid Preparation, QC and Library building for Next Generation Sequencing

Presenters: Dr Etienne Slabbert and Dr Stanton Hector

Duration: 3 Days

The focus of the workshop will be the preparation and quality control (QC) of nucleic acids (DNA and RNA) for next-generation sequencing applications. During the workshop basic DNA and RNA extraction theory will be presented which will be followed by practical sessions. The nucleic acids extracted during the practical sessions will be quantified and QC’ed using the NanoDrop ND-1000 and the Qubit 2.0 fluorometer. Further QC (integrity) will be performed using a real-time based qPCR assay (DNA) and the Agilent 2100 BioAnalyzer (pro- and eukaryotic RNA). This will be followed with theoretical sessions on library preparation for next-generation sequencing (Ion Torrent). A hands-on session for quantifying and assessing the quality of NGS-libraries will follow. The workshop will conclude with some basic analysis and interpretation of NGS-data as generated by the Ion Torrent platforms.

Human Movement Analysis

11. 3D Neuro-biomechanical Analysis (Tygerberg Campus)

Presenter: Dr John Cockcroft

Duration: 2 Days

In this workshop participants will be trained to operate a synchronized motion capture system (Vicon), force-plate system (Bertec) and wireless EMG system (Noraxon) to perform neuro-biomechanical testing and analysis of human movement.

Day 1: Data collection

  • Morning session: demonstration
    • System setup (camera calibrations, database management)
    • Subject preparation (model calibration, anthropometrics)
    • Test protocols (procedures for capturing, signal quality checks)
  • Afternoon practical session: capturing different functional tasks

Day 2: Data analysis

  • Morning session
    • Data pre-processing (marker reconstruction, filtering)
    • Musculoskeletal modelling (processing pipelines, sources of error)
    • Data segmentation techniques (event detection, time-normalization)
  • Afternoon practical session: processing and exporting data