Today HOUSEKEEPING, background, presentations DNA extraction from snails DNA extraction from...
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Transcript of Today HOUSEKEEPING, background, presentations DNA extraction from snails DNA extraction from...
Today• HOUSEKEEPING,
background, presentations• DNA extraction from snails• DNA extraction from parasites (cercariae)• Start protocols (timing!)• Background on methods• Complete extractions protocol
PARASITES AND SNAILPARASITES AND SNAIL BIOLOGY BIOLOGY
“identity, possibilities”phylogenetics
“intentions”transcriptomics
PCRrDNA/mito
BioanalyzerDNA-free,
direct sequencing
gel electrophoresisnanodrop spec
Sequence ID (BLAST)editing
Phylogenetics
electrophoresisRT-PCR
gel
CTAB/DNAzol
Trizol
TA cloning, B/W screening
M13 sequencing
Primer design, walking
Qiagen plasmid extraction Restriction digests
DNA
RNA
GenBank submission
Longitude 106°35'54.52"W(Google Earth)
Physid
Shady Lakes: SNAILS AND PARASITES
Lattitude 35°12'59.15"N
Specimensinfected snails +released cercariaefixed in 80% EtOH
Physella sp?
Wethington Leydeard,2007
Things to be careful about with DNA Extraction
• Safety • Vortexing/pipetting• Too much starting material?• When is a good time to stop• Contamination• Add too much of a reagent? Don’t freak out • What should you use to reconstitute or dissolve your
DNA?• Where is the DNA?• Solution of DNA too concentrated/too dilute
1 SP1/PP1
2(3) SP2/PP2
SAMPLE ASSIGNMENTS
9SP1/PP1
10SP2/PP2
5SP5/PP5
6SP4/PP4
8SP4/PP4
7SP5/PP5
EXTRACT DNA
HANDOUT 3 complete step 1-6 of DNA extraction
15 minute powerpoint topics • Discovery of DNA structure• Restriction enzymes• Southern blotting• Cloning • The first sequenced gene• PCR, specificity and sensitivity• RAPDs• q-PCR• BAC libraries• ESTs• BLAST and database searches• Microarrays • Forensics • Genome sequencing , the $1000 genome• Next generation sequencing• Bioinformatics• Epigenetics• "non-coding" RNA• C-value paradox• Phylogenetic genomics• Archeological genomics• YOUR favorite gene (check with instructor)
Research your topic
(Coen provides guidance on literature)
Prepare, present ppt presentation on topic (12 + 3 format)
Participate in Q and A
Start Sept 22nd
Sample Preservation• Collect from the field, freeze at -70C
– alternatives• Extraction buffer - good DNA, unhappy airlines• RNA later (AMBION) - good RNA (DNA)• 70-100% ethanol - dehydrates DNA• Guanidinium isothiocyanate -good for RNA• Dry - varying results (good/low yield/degradation) • Formaldehyde - Bad: cross-links (formic acid, depurinates DNA (A,G)
DNA/RNA WHERE?
DNA
(shell)tissuecellsnucleus/mitochondria
Also in the way, lipids, mucopolysaccharides, proteins (enzymes)Avoid all that and obtain DNA/RNA
RNA(mechanicaldisruption)
parasites/other symbionts, animal, plant, bacterial, fungal?
DNA ExtractionCTAB-cetyltrimethylammonium bromide, cationic detergent,
forms complexes with (poly)saccharides (and protein?) to help mucopolysaccharide removal
EDTA-ethylene di-amino tetra acetic acid,chelates divalent metals, cofactors for nucleases
Tris/HCl pH 8.0 - maintains pH (important for DNA)Beta mercapto ethanol - lyses cells, denatures proteinsProteinase K- degrades proteins (at 60C!)NaCl - helps precipitationHigh temperature - inactivates enzymes
DNA isolation• Industry provides BLACK BOX
• SDS (detergent) dissolves lipids• Chaotropic salts denature proteins, disrupt protein structure,
cluster nucleic acids
A chaotropic agent, also known as chaotropic reagent and chaotrope, is a substance which disrupts the three dimensional structure in macromolecules such as proteins, DNA, or RNA and denatures them.Chaotropic agents interfere with stabilizing intra-molecular interactions mediated by non-covalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic effects.
Chaotropic reagents include:
Urea
Guanidinium chloride
http://en.wikipedia.org/wiki/Chaotropic_agent
http://www.mrcgene.com/dnazoffer.htm
Organic (Chloroform) Extraction
• Mix snail extract with chloroform,centrifuge to separate into phases.
DNA (>pH8.0)RNA proteins
chloroformdebris, other
Alcohol precipitation
Alcohol plus aqueous (UPPER) phaseDNA cannot retain water in the presence of
alcohol, salt and will precipitate outHow it works physico-chemically, see http://bitesizebio.com/253/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/
EtOH (100-95%) or isopropanol (100%)Clean-up and dissolve in molecular grade H2O
DNA and RNA• DNA from nucleus AND mitochondria (identity and genomics)• Organic extraction needs pH 8 or higher• (+ RNA) RNA transcribed from genes
• RNA cytosol (intentions and regulation)• Also at < pH 8.0• (+ DNA) different in structure, susceptible to break down,
degradation.NEW kits greatly facilitate working with RNA, RNA later, RNAseZAP.
NUCLEIC ACIDS QUALITY?
• See fibers, pellets? Or not?
• Quality, Amount, Composition?
• Gelelectrophoresis• Spectrophotometry