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Short course on DNA barcoding methods November 29, 2011

Short course on DNA barcoding methods November 29, 2011. DNA Extraction. Darío Lijtmaer Museo Argentino de Ciencias Naturales “Bernardino Rivadavia ”. Organization of the talk. 1) Alternatives available to a person/lab interested in DNA barcoding. 2) Equipment needed for DNA extraction.

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Short course on DNA barcoding methods November 29, 2011

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  1. Short course on DNA barcoding methods November 29, 2011 • DNA Extraction Darío Lijtmaer MuseoArgentino de CienciasNaturales “Bernardino Rivadavia”

  2. Organization of the talk 1) Alternatives available to a person/lab interested in DNA barcoding. 2) Equipment needed for DNA extraction. 3) Overview of extraction protocols. 4) Minimizing the risks of contamination. 5) Storing of DNA extracts. 6) Discussion and questions.

  3. Alternatives available to a person/lab interested in DNA barcoding a) Send/take tissue sub-samples for processing at a high throughput facility (different types of collaborations are possible).

  4. Alternatives available to a person/lab interested in DNA barcoding a) Send/take tissue sub-samples for processing at a high throughput facility (different types of collaborations are possible). Both groups can be involved in the whole study... Barcodes obtained at the CCDB Field work in Argentina (birds) Tissues and vouchers deposited at MACN Argentinian students take subsamples and are trained at the CCDB Analysis performed together

  5. Alternatives available to a person/lab interested in DNA barcoding a) Send/take tissue sub-samples for processing at a high throughput facility (different types of collaborations are possible). b) Perform the first laboratory steps of the barcoding process (extraction and amplification) in your lab and send the PCR products for sequencing.

  6. Alternatives available to a person/lab interested in DNA barcoding a) Send/take tissue sub-samples for processing at a high throughput facility (different types of collaborations are possible). b) Perform the first laboratory steps of the barcoding process (extraction and amplification) in your lab and send the PCR products for sequencing. PCR products sequenced at CCDB Students trained Tissues and vouchers deposited at MACN and other institutions Field work in Argentina and neighbouring countries Samples processed at MACN (small and medium scale) Analysis performed together

  7. Alternatives available to a person/lab interested in DNA barcoding a) Send/take tissue sub-samples for processing at a high throughput facility (different types of collaborations are possible). b) Perform the first laboratory steps of the barcoding process (extraction and amplification) in your lab and send the PCR products for sequencing. c) Perform the entire process in your lab.

  8. Equipment: basic for a small-sized facility • Hundreds or few thousands of barcodes produced per year. • Tube scale. Water bath or incubator Centrifuge Vortex Pipettes Scale Disposables and reagents Autoclave

  9. Equipment: medium-sized facility • Up to 20,000 thousand barcodes produced per year. • Plate scale. Plate centrifuge Incubator Scale Disposables and reagents Pipettes Autoclave

  10. Equipment: high-throughput facility • Up to hundreds of thousands of barcodes produced per year. • Plate scale, robotic protocols. • All pieces of equipment mentioned above plus... Robots DNA extractors Sequencing machines

  11. Overview of extraction protocols None of the lab protocols/procedures are necessarily different from those used for other mitochondrial markers or other projects.

  12. Overview of extraction protocols None of the lab protocols/procedures are necessarily different from those used for other mitochondrial markers or other projects. However... a) Due to the scale of the project efforts are made to reduce the cost of the molecular steps of the pipeline. b) Certain requirements are needed to achieve the barcode data standard. As a consequence innovations and development of new, more efficient protocols/proceedures are frequent in the context of the project.

  13. Overview of extraction protocols

  14. Overview of extraction protocols

  15. Overview of extraction protocols: CCDB • Versions of the protocol: • Manual with individual tubes in small-sized facilities. • Manual with 96 well plates in medium-sized facilities. • Robotic with 96 well plates in high-throughput facilities. It can be used for vertebrates and most invertebrates. Contrary to intuition, using bigger sample fragments is not better!! www.barcodeoflife.org

  16. Overview of extraction protocols: CCDB There is a similar extraction protocol developed for plants, which is also used for fungi, mollusks and echinoderms

  17. Overview of extraction protocols In the case of very small invertebrates, in which the whole specimen has to be used for DNA extraction, one option is to use a protocol that allows the recovery of the exoskeleton as a vouchers.

  18. Minimizing the risk of contamination General practices Clean workspace and sterile tips, tubes, etc.

  19. Minimizing the risk of contamination General practices Clean workspace and sterile tips, tubes, etc. Clean tweezers between samples (ELIMINase in vertebrates, ethanol or burning in invertebrates).

  20. Minimizing the risk of contamination General practices Clean workspace and sterile tips, tubes, etc. Clean tweezers between samples (ELIMINase in vertebrates, ethanol or burning in invertebrates). If working with plates, cover all the rows of wells with caps except the one you are working on and avoid transferring tissue above the plate.

  21. Minimizing the risk of contamination General practices Clean workspace and sterile tips, tubes, etc. Clean tweezers between samples (ELIMINase in vertebrates, ethanol or burning in invertebrates). If working with plates, cover all the rows of wells with caps except the one you are working on and avoid transferring tissue above the plate. Three sets of pipettes: one for extraction, one for preparing PCR and one for PCR products (for example for gel loading).

  22. Minimizing the risk of contamination General practices Clean workspace and sterile tips, tubes, etc. Clean tweezers between samples (ELIMINase in vertebrates, ethanol or burning in invertebrates). If working with plates, cover all the rows of wells with caps except the one you are working on and avoid transferring tissue above the plate. Three sets of pipettes: one for extraction, one for preparing PCR and one for PCR products (for example for gel loading). If working with difficult samples, such as degraded DNA... Special laboratory design (for example two separate doors that are opened in sequence, presence of UV light). Be extra-careful (for example, change gloves more often).

  23. Storage of DNA extracts Alternatives available for long-term storage of DNA extracts Frozen extracts Ultracold freezer or Liquid Nitrogen Room temperature Dried extracts on ceramic beads or FTA paper

  24. Questions and discussion Thank you very much!

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