Welcome !!!

1. Welcome !!! FISH 543 / OCEAN 575 Molecular Techniques

2. Danielle Mitchell Room MAR 175 mitcheld@u.washington.edu Office hours: By appointment Dr Lorenz Hauser Room MAR 207 Tel: 685-3270 lhauser@u.washington.edu Office hours: By appointment Introductions

3. MMBLMarine Molecular Biotechnology Lab • 4 faculty • 2 fisheries (Naish, Hauser) • 2 oceanography (Armbrust, Rocap) • Main research areas • Conservation Genetics • Molecular Ecology • Genomics • Phytoplankton Ecology • Come and talk to people! We are here MMBL

4. Introduce yourself • Name • Home Department • Project • Background • Specific question • Molecular methods

5. Pairs • Similar projects • Can share some tasks • buffers, extraction, cloning etc. • Lab today • Suggested pairs • Jim Franks – Pilar Montepan • Brian Kristall – Gang Xin • Kristi Straus – Nick Adams • Thomas Unfried – Bill Webb • Danny Garrett – Alfred Sidman

6. Some General Info • Prerequisite • FISH 542 / OCEAN 574 • Check web page • Username – Ocean574 • Password - Gryffindor • Will present some information • Read! • Hillis et al. (1996) Molecular Systematics. Sinauer • References on 542 homepage • Ask • Textbook • Guidebook ‘Maniatis’ • Sambrook & Russell et al. 2001 • Copy in lab – LEAVE IT THERE!!

7. Communication • Lab meeting • Tuesdays 1:30 • Ask questions • In labs • Arrange meetings • E-mail • To whole class • To us • Talk to each other • Class mates • Students in MMBL • Check the homepage frequently • Subject to change • Uploaded: • Lecture slides • Protocols of general interest • Links • Papers • Contribute • Links • Protocols

8. Course description • Lab Meetings • Facilities • Main lab • FTR 129 • Meetings • FTR 103 • Some equipment • MMBL tour • Initial training • Set lab exercises • Designed to show general procedures • Not only own project • Lab access • Two sessions • Tuesdays & Thursdays • Open access other times • Recommended to finish projects • During building opening hours • Will get keycode lock

9. Facilities • FTR 129 • Main lab • Bench space, extraction, electrophoresis etc. • FTR 103 • Meetings • Food and drink • MMBL • Marine Studies Building • Some equipment • Sequencer • Gel scanner • Emergency supplies • Let us know! • Computers • Three in the lab • More PCs in FSH 207 & FSH 209 • Macs • ask • Software links on webpage

10. Safety • No eating or drinking in lab • Use FTR 103 • Wear labcoats and gloves • Goggles and masks for some materials • No open shoes • Assume all materials are hazardous • Many are • If in doubt, consult MSDS • links on webpage • Particularly nasty stuff will be flagged • Some on webpage • Spillages • Report to instructors • Electrophoresis • Switch off before touching • If in doubt -ask us

11. Assessment • Detailed description on homepage • Proposal (10%) • 5 pages • Example on web • Due end of next week • Friday Jan 16, 2004 • Proposal review • Two colleagues • Be nice but critical • Instructor • Due end of week 3 • Friday Jan 23, 2004

12. Assessment • Lab Notebook • Essential part of the course • Reproducibility • Can stick in protocols • Provide details and describe deviations • Use bound lab notebooks • Ring binders lose leaves • Will collect lab notebooks 2-3 times during the quarter • Lab Participation • Show up! • Tuesdays & Thursdays • Try hard! • Discussion participation • Will meet every Tuesday

13. Assessment • Presentation • Meeting in final week • Tuesday March 17 • Invite MMBLers • Present results • 10 min • Future work • Report • 8 pages • Incorporate comments on talk and proposal • Scientific paper format • Introduction • Methods • Results • Discussion • Due Thursday March 19

14. The proposal • Needed because FISH 542 / OCEAN 574 not offered • Main Aims • Sort out ideas • Based on background • Define questions • Check feasibility • Timeline • Wider significance • Dissertation projects • Can be used (not verbatim if it’s not yours) • Concentrate on work in class • Will be considered in assessment • Budget • Idea of how much it is • Estimate of additional funds required

15. Sample collection and storage; DNA extraction and storage

16. Tissue collection • Almost any living tissue • Preferably fresh organisms • Less stringent (ancient DNA) • Quantity less important • Can get DNA out of single cells • more important is ratio tissue / preservative • Non-destructive sampling • feathers, hairs, feces • Contain cells or cell debris

17. Tissue preservation • DNA • inhibit enzyme activity of enzymes eating DNA • Usually accomplished by dehydration or freezing • alcohol, high salt concentration or drying • Alcohol is most commonly used technique • Small material (cells): frozen in TE buffer • Formalin preservation is no good • Preferred method in museums • causes degradation and irreversible cross-linking. • Some protocols are at http://www.public.iastate.edu/~curteck/Formalin_Fixed_DNA.htm

18. Tissue storage • Important issue • Reproducing results • Temporal changes • Endangered species • Three main ways • Freezing • Burke museum genetic specimen collection • Problem: equipment or power failure • Ethanol • Keep below room temperature • Explosive ! • Drying • Herbarium specimen • DNA is surprisingly stable • Extracted from very old specimen • But – only some markers can be applied

19. DNA extraction – the basic concept Chemical Enzymatic Mechanic • SDS, sarcosyl • CTAB • Proteinase K • Lysozyme • Freezing • Sonication • Grinding Cell lysis Organic solvents Salt DNA binding • Phenol • chloroform • Sodium chloride • Sodium acetate • Membrane • Beads Protein removal Alcohol • Ethanol • Iso-propanol DNA precipitation Process Common procedure

20. Many variations on the theme • Depending on DNA quality and throughput required • Problem often degradation • DNA in small fragments • Most PCR based methods get away with bad DNA • Hotshot (Biotechniques 29:52 (2000)) • Boil it and PCR it • Salting out • But often PCR inhibitors • Co-purify with DNA • humic acids • Secondary cell components in plants • Can be tested by adding extract to working PCR • Ways to further purify DNA • Ultracentrifugation in CsCl gradients • Gel electrophoresis • Chromatography • Rapid development of methods • Talk to people • Newsgroups • Company newsletters

21. Commercial kits • Bind DNA selectively to a membrane • Wash rest away • Many specific commercial kits • Plant • Animals • Soil • Feces

22. DNA storage • Difficult but important field • DNA techniques only in 30 years • Little experience with long term storage • Development of new markers • Verify results • Compare markers • Temporal studies • Endangered species • Climate change • Anthropogenetic change • DNA is very stable • May survive up to 100,000 years • Drosophila paper (Colton & Clark 2001) • Six different storage methods (2 years) • Three extraction methods • PCR of 800 bp fragment •  it ain’t matter • Lots of reports of degraded, unusable DNA

23. DNA storage • Main options • Salt solution • EDTA – prevents enzyme activity • Only short term • Concentrated salt • DMSO • Freezing • expensive • risky • Alcohol • Explosive • Needs regular checking (evaporation) • degradation • Store at cold temperature (4oC, -20oC) • Dried • Extract and purify • Apply to membranes • Store dry • Make sure it’s dry

24. Summary Mr. DNA • Tissue collection • Almost any tissue • Preferably fresh • Fresh tissue for allozymes • Tissue storage • Aim: dehydration • Main methods: freezing, alcohol, salt, drying • DNA extraction • Central theme: cell lysis, protein removal, DNA precipitation • Many variations • Depending on quality required, throughput and costs • Commercial kits • Columns or beads • DNA storage • Freezing, alcohol, salt, drying

25. Laboratory • Part A – pipetting • Intro to pipettes • Some exercises • Part B – Making buffers • Required for DNA extraction • Work in pairs • Part C – Tour of MMBL • See facilities • Part D – set up digestions for Thursday • Two methods • Salt • Qiagen DNeasy kit