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