1. Today HOUSEKEEPING, background, presentations DNA extraction from snails DNA extraction from parasites (cercariae) Start protocols (timing!) Background on methods Complete extractions protocol

2. PARASITES AND SNAIL BIOLOGY “identity, possibilities” phylogenetics “intentions” transcriptomics PCR rDNA/mito Bioanalyzer DNA-free, direct sequencing gel electrophoresis nanodrop spec Sequence ID (BLAST) editing Phylogenetics electrophoresis RT-PCR gel CTAB/DNAzol Trizol TA cloning, B/W screening M13 sequencing Primer design, walking Qiagen plasmid extraction Restriction digests DNA RNA GenBank submission

3. Longitude 106°35'54.52"W (Google Earth) Physid Shady Lakes: SNAILS AND PARASITES Lattitude 35°12'59.15"N Specimens infected snails + released cercariae fixed in 80% EtOH Physella sp? Wethington Leydeard, 2007

4. 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

5. 1 SP1/PP1 2(3) SP2/PP2 SAMPLE ASSIGNMENTS 9 SP1/PP1 10 SP2/PP2 5 SP5/PP5 6 SP4/PP4 8 SP4/PP4 7 SP5/PP5

6. EXTRACT DNA HANDOUT 3 complete step 1-6 of DNA extraction

7. 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

8. 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)

9. DNA/RNA WHERE? DNA (shell) tissue cells nucleus/mitochondria Also in the way, lipids, mucopolysaccharides, proteins (enzymes) Avoid all that and obtain DNA/RNA RNA (mechanical disruption) parasites/other symbionts, animal, plant, bacterial, fungal?

10. DNA Extraction CTAB-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 proteins Proteinase K- degrades proteins (at 60C!) NaCl - helps precipitation High temperature - inactivates enzymes

11. DNA isolation Industry provides BLACK BOX SDS (detergent) dissolves lipids Chaotropic salts denature proteins, disrupt protein structure, cluster nucleic acids

12. 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

13. Organic (Chloroform) Extraction Mix snail extract with chloroform, centrifuge to separate into phases. DNA (>pH8.0) RNA proteins chloroform debris, other

14. Alcohol precipitation Alcohol plus aqueous (UPPER) phase DNA cannot retain water in the presence of alcohol, salt and will precipitate out How 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 H 2 O

15. 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.

16. NUCLEIC ACIDS QUALITY? See fibers, pellets? Or not? Quality, Amount, Composition? Gelelectrophoresis Spectrophotometry