FISH%20543%20/%20OCEAN%20575

1
Welcome !!!

• FISH 543 / OCEAN 575
• Molecular Techniques

2
Introductions

• Danielle Mitchell
• Room MAR 175
• mitcheld_at_u.washington.edu
• Office hours By appointment

• Dr Lorenz Hauser
• Room MAR 207
• Tel [phone]
• lhauser_at_u.washington.edu
• Office hours By appointment

3
MMBL Marine 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!!

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Communication

• Check the homepage frequently
• Subject to change
• Uploaded
• Lecture slides
• Protocols of general interest
• Links
• Papers
• Contribute
• Links
• Protocols

• Lab meeting
• Tuesdays 130
• Ask questions
• In labs
• Arrange meetings
• E-mail
• To whole class
• To us
• Talk to each other
• Class mates
• Students in MMBL

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Course description

• 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

• Lab Meetings
• Facilities
• Main lab
• FTR 129
• Meetings
• FTR 103
• Some equipment
• MMBL tour

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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

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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

Item
Research Proposal 10
Proposal Review 10
Lab Notebooks 20
Lab Participation 15
Discussion Participation 15
Presentation 10
Report 20
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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

Item
Research Proposal 10
Proposal Review 10
Lab Notebooks 20
Lab Participation 15
Discussion Participation 15
Presentation 10
Report 20
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

Item
Research Proposal 10
Proposal Review 10
Lab Notebooks 20
Lab Participation 15
Discussion Participation 15
Presentation 10
Report 20
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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 its 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
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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

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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.ed
  u/curteck/Formalin_Fixed_DNA.htm

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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

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DNA extraction the basic concept
Process
Common procedure
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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 2952 (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

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Commercial kits

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

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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 aint matter
• Lots of reports of degraded, unusable DNA

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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 its dry

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Summary

• 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

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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