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Marine Invertebrates Dirk Steinke

Marine Invertebrates Dirk Steinke. Short Course on DNA Barcoding Methods 29 Nov 2011. Preservation. The good Ethanol preserved, not older than 5 years frozen tissue RNA later. The bad Ethanol preserved, older than 10 years at room temp DMSO (cross-reacts with Ethanol)

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Marine Invertebrates Dirk Steinke

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  1. Marine Invertebrates Dirk Steinke Short Course on DNA Barcoding Methods 29 Nov 2011

  2. Preservation • The good • Ethanol preserved, not older than 5 years • frozen tissue • RNA later • The bad • Ethanol preserved, older than 10 years at room temp • DMSO (cross-reacts with Ethanol) • Isopropanol • The ugly • formalin • spirit • …

  3. Sampling • mtDNA rich tissue where possible • muscle tissue (larger animals e.g. fish) • legs (arthropods) • tube feet (echinoderms) The smaller the better!

  4. Extraction Regular kits will do in many cases, but Echinoderms Polychaetes Mollusks work much better with CTAB extraction protocols (binds to polysaccharides that can inhibit PCR)

  5. PCR Taxon-specific primers are key Check out http://connect.barcodeoflife.net/group/marinebarcoding

  6. PCR Taxon specific primers needed Majority works with one primer pair or cocktail • Fish • Cephalopoda1 • Gastropoda • Pycnogonida • Echinodermata • Brachipoda • Crustacea • Annelida • Cnidaria • Porifera Notoriously difficult • Foraminifera • Bivalvia2 • Tunicata 1some families have a tandem copy of COI 2two different COI-versions (male/female)

  7. Sequencing Multiplex primer cocktails 5’ 3’ M13-tail • M13 tail for sequencing multiplex products • M13 also useful for standard primer pairs (Folmer-tailed version very successful)

  8. Editing • some groups exhibit indels more frequently (e.g. crustacea, mollusks) • watch out for pseudogenes (often easy to spot through stop codons) • proper alignment is crucial • some symbiotic bacteria can be amplified using universal primers

  9. Thank you!

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