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An overview of DNA extraction protocols from small scale labs right up to high-throughput facilities.

An overview of DNA extraction protocols from small scale labs right up to high-throughput facilities.

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  • 1. Short course on DNA barcoding methods November 29, 2011 DNA Extraction Darío LijtmaerMuseo Argentino de Ciencias Naturales “Bernardino Rivadavia”
  • 2. Organization of the talk1) 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 barcodinga) Send/take tissue sub-samples for processing at a high throughputfacility (different types of collaborations are possible).
  • 4. Alternatives available to a person/lab interested in DNA barcodinga) Send/take tissue sub-samples for processing at a high throughputfacility (different types of collaborations are possible).Both groups can be involved in the whole study... BarcodesField work in Tissues and Argentinian obtained at the Argentina vouchers students take CCDB (birds) deposited at subsamples MACN and are trained at the CCDB Analysis performed together
  • 5. Alternatives available to a person/lab interested in DNA barcodinga) Send/take tissue sub-samples for processing at a high throughputfacility (different types of collaborations are possible).b) Perform the first laboratory steps of the barcoding process (extractionand 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. Students PCR products trained sequenced at CCDB Tissues andField work in vouchersArgentina and deposited atneighbouring MACN and other Samples countries institutions processed at MACN (small and Analysis performed medium scale) together
  • 7. Alternatives available to a person/lab interested in DNA barcodinga) Send/take tissue sub-samples for processing at a high throughputfacility (different types of collaborations are possible).b) Perform the first laboratory steps of the barcoding process (extractionand 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. Incubator Plate centrifuge Pipettes Scale Disposables and reagents 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... DNA extractors Robots Sequencing machines
  • 11. Overview of extraction protocolsNone of the lab protocols/procedures are necessarily different from thoseused for other mitochondrial markers or other projects.
  • 12. Overview of extraction protocolsNone of the lab protocols/procedures are necessarily different from thoseused for other mitochondrial markers or other projects.However...a) Due to the scale of the project efforts are made to reduce the cost ofthe 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 efficientprotocols/proceedures are frequent in the context of the project.
  • 13. Overview of extraction protocols Approximate costMethods DNA quality (per sample, USD)Chelex - Ethanol and Salt 0.35 poorCommercial kits (Genelute Mammalian kit) 2.00 high
  • 14. Overview of extraction protocols Approximate costMethods DNA quality (per sample, USD)Chelex - Ethanol and Salt 0.35 poorCommercial kits (Genelute Mammalian kit) 2.00 high“Home made” glass fiber extraction 0.50 high
  • 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: CCDBThere is a similar extraction protocol developed for plants, which is alsoused for fungi, mollusks and echinoderms
  • 17. Overview of extraction protocolsIn the case of very small invertebrates, in which the whole specimen hasto be used for DNA extraction, one option is to use a protocol that allowsthe recovery of the exoskeleton as a vouchers.
  • 18. Minimizing the risk of contaminationGeneral practices Clean workspace and sterile tips, tubes, etc.
  • 19. Minimizing the risk of contaminationGeneral practices Clean workspace and sterile tips, tubes, etc. Clean tweezers between samples (ELIMINase in vertebrates, ethanol orburning in invertebrates).
  • 20. Minimizing the risk of contaminationGeneral practices Clean workspace and sterile tips, tubes, etc. Clean tweezers between samples (ELIMINase in vertebrates, ethanol orburning in invertebrates). If working with plates, cover all the rows of wells with caps except theone you are working on and avoid transferring tissue above the plate.
  • 21. Minimizing the risk of contaminationGeneral practices Clean workspace and sterile tips, tubes, etc. Clean tweezers between samples (ELIMINase in vertebrates, ethanol orburning in invertebrates). If working with plates, cover all the rows of wells with caps except theone you are working on and avoid transferring tissue above the plate. Three sets of pipettes: one for extraction, one for preparing PCR andone for PCR products (for example for gel loading).
  • 22. Minimizing the risk of contaminationGeneral practices Clean workspace and sterile tips, tubes, etc. Clean tweezers between samples (ELIMINase in vertebrates, ethanol orburning in invertebrates). If working with plates, cover all the rows of wells with caps except theone you are working on and avoid transferring tissue above the plate. Three sets of pipettes: one for extraction, one for preparing PCR andone 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 areopened 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 Room temperature Ultracold freezer or Dried extracts on Liquid Nitrogen ceramic beads or FTA paper
  • 24. Questions and discussionThank you very much!