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