Dario Lijtmaer - PCR Amplification

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Discussing the equipment, methods/protocols, PCR product verification and shipment of PCR products

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Dario Lijtmaer - PCR Amplification

  1. 1. Short course on DNA barcoding methods November 29, 2011 PCR amplification Darío LijtmaerMuseo Argentino de Ciencias Naturales “Bernardino Rivadavia”
  2. 2. Organization of the talk1) Equipment needed for PCR amplification.2) Overview of PCR protocols.3) Product verification: agarose gels.4) Minimizing the risks of contamination.5) Shipping and storing DNA extracts.6) Discussion and questions.
  3. 3. Equipment: basic for a small-sized facility- Hundreds or few thousands of barcodes produced per year.- Tube scale. Incubator Autoclave Thermocycler Pipettes Disposables and reagents Vortex
  4. 4. Equipment: medium-sized and high throughput facilities- Many thousand barcodes produced per year.- Plate scale. Incubator Autoclave Thermocycler Pipettes Disposables and reagents Vortex
  5. 5. Overview of amplification protocolsNone of the lab protocols/procedures are necessarily different from thoseused for other mitochondrial markers or other projects.
  6. 6. Overview of amplification 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 (e.g. small PCR volumes).b) Certain requirements are needed to achieve the barcode data standard(e.g. minimum length).As a consequence innovations and development of new, more efficientprotocols/proceedures are frequent in the context of the project.
  7. 7. Overview of amplification protocols: CCDB Animals: COI This protocol can be used with: • Individual tubes in small-sized facilities. • 96 well plates in medium-sized or high throughput facilities. www.barcodeoflife.org
  8. 8. Overview of amplification protocols: CCDB Plants and fungi This protocol can be used with: • Individual tubes in small-sized facilities. • 96 well plates in medium-sized or high throughput facilities. www.barcodeoflife.org
  9. 9. Overview of amplification protocols: PCR mix (CCDB) Small PCR volumes: 12.5 ml for most reactions (e.g. COI in animals andrbcL in plants), 6.25 ml for matK (plants). • Cost-efficient.
  10. 10. Overview of amplification protocols: PCR mix (CCDB) Small PCR volumes: 12.5 ml for most reactions (e.g. COI in animals andrbcL in plants), 6.25 ml for matK (plants). • Cost-efficient. Trehalose is used as part of the PCR mix. • It allows freezing aliquots of the mix (useful for high throughput facilities). • It estabilizes the reaction.
  11. 11. Overview of amplification protocols: PCR mix (CCDB) Small PCR volumes: 12.5 ml for most reactions (e.g. COI in animals andrbcL in plants), 6.25 ml for matK (plants). • Cost-efficient. Trehalose is used as part of the PCR mix. • It allows freezing aliquots of the mix (useful for high throughput facilities). • It estabilizes the reaction. Platinum taq polymerase. • High success and band intensity, less optimization needed. • Hot start . • Stable at room temperature.
  12. 12. Overview of amplification protocols: PCR mix (other tips) BSA can be added to the PCR mix to improve PCR results. This is done atSmithsonian LAB with invertebrate samples and in the African Centre forDNA Barcoding with plant samples.
  13. 13. Overview of amplification protocols: PCR mix (other tips) BSA can be added to the PCR mix to improve PCR results. This is done atSmithsonian LAB with invertebrate samples and in the African Centre forDNA Barcoding with plant samples. DMSO can also be added to improve PCR results with difficult samples.
  14. 14. Overview of amplification protocols: primersPrimer choice is a key aspect of PCR success and probably the only aspectof amplification that is taxon-dependent.
  15. 15. Overview of amplification protocols: primersPrimer choice is a key aspect of PCR success and probably the only aspectof amplification that is taxon-dependent.Ideal situation: universal primers.Real world: various sets of primers are to be used (and sometimescombined) depending on the taxonomic group. There is also more thanone option for each group.
  16. 16. Overview of amplification protocols: primersPrimer choice is a key aspect of PCR success and probably the only aspectof amplification that is taxon-dependent.Ideal situation: universal primers.Real world: various sets of primers are to be used (and sometimescombined) depending on the taxonomic group. There is also more thanone option for each group.
  17. 17. Overview of amplification protocols: primersPrimer choice is a key aspect of PCR success and probably the only aspectof amplification that is taxon-dependent.Ideal situation: universal primers.Real world: various sets of primers are to be used (and sometimescombined) depending on the taxonomic group. There is also more thanone option for each group.We included as part of the complementary materials: the list of primers that are used at the CCDB with each taxonomicgroup, the sequence of those primers and the thermocycling program thatis used with each primer. the list of primers and the thermocycling program used at theSmithsonian LAB.
  18. 18. Overview of amplification protocolsPCR protocols also depend on the quality of the samples used.
  19. 19. Overview of amplification protocolsPCR protocols also depend on the quality of the samples used.For example, samples with potentially degraded DNA, such as relativelyold museum samples, require special primers designed to amplifyshorter, overlapping fragments (e.g. 200 bp long).
  20. 20. Overview of amplification protocolsPCR protocols also depend on the quality of the samples used.For example, samples with potentially degraded DNA, such as relativelyold museum samples, require special primers designed to amplifyshorter, overlapping fragments (e.g. 200 bp long).
  21. 21. Product verification: agarose gelsPCR results are usually visualized in agarose gels.Depending on the scale (and funding) home made gels or pre-cast gels areused.
  22. 22. Product verification: agarose gelsPCR results are usually visualized in agarose gels.Depending on the scale (and funding) home made gels or pre-cast gels areused.For plate-scales (medium scale or high-throughput) usually a threshold isestablished (e.g. 75/95 bands at CCDB) and when a plate results are abovethe threshold the entire plate is sequenced .
  23. 23. Mailing PCR productsIf sequencing is not done on-site, PCR products are transferred to a platecontaining trehalose and dried before mailing them to a high throughputfacility.
  24. 24. Minimizing the risk of contaminationGeneral practices Clean workspace and sterile tips, tubes, etc.
  25. 25. Minimizing the risk of contaminationGeneral practices Clean workspace and sterile tips, tubes, etc. Three sets of pipettes: one for extraction, one for preparing PCR andone for PCR products (for example for gel loading).
  26. 26. Minimizing the risk of contaminationGeneral practices Clean workspace and sterile tips, tubes, etc. 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).
  27. 27. Q&A and discussion Thank you very much!

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