welcome !!! fish 543 / ocean 575 molecular techniques
Post on 21-Dec-2015
219 views
TRANSCRIPT
Welcome !!!FISH 543 / OCEAN 575
Molecular Techniques
Introductions
• Danielle Mitchell– Room MAR 175
– Office hours: By appointment
• Dr Lorenz Hauser– Room MAR 207
– Tel: 685-3270
– Office hours: By appointment
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
hereMMBL
Introduce yourself
• Name• Home Department• Project
– Background
– Specific question
– Molecular methods
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
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!!
Communication
• Check the homepage frequently– Subject to change
– Uploaded:• Lecture slides
• Protocols of general interest
• Links
• Papers
– Contribute• Links
• Protocols
• 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
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
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
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
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
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
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
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
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
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
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
DNA extraction – the basic concept
Cell lysis
Protein removal
DNA precipitation
Process Common procedure
•SDS, sarcosyl•CTAB
•Proteinase K•Lysozyme
•Freezing•Sonication•Grinding
Chemical
Enzymatic
Mechanic
Alcohol •Ethanol•Iso-propanol
•Phenol•chloroform
•Sodium chloride•Sodium acetate
•Membrane•Beads
Organic solvents
Salt
DNA binding
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
Commercial kits
• Bind DNA selectively to a membrane
• Wash rest away
• Many specific commercial kits– Plant– Animals– Soil– Feces
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
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
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
Mr. DNA