Today

1. Today • HOUSEKEEPING presentations • DNA extraction from snails/parasites • Start protocols (timing!) • Homework • Background on methods • Complete extractions protocol

2. ALSO TOPICS: 1st DNA discovery | talk to me wednesday 4-5/7 Sep 2nd Southern blotting | (out 6 Sep)

3. TOPICS.ppt 1 DNA discovery – Katerina Zlatkin 2 Restriction Enzymes – Nicole Caroll 3 Southern Blotting – Jonathan Locke 4 Cloning – Kimberly Wright 5 First sequencing methods/gene – Jessie Marlenee 6 BAC Libraries – Alan Buser 7 PCR – Katherine Graf 8 ESTs – John McCready 9 BLAST/GenBank – Zach Potts 10 microarrays – Vincent Cutillas 11 qPCR – Taylor Britton 12 Mitogenomics – Joe Medley 13 Genome sequencing – Ashlynn Bennett 14 forensics – Dante Trujillo 15 NGS – Pankoj Kumar Das 16 bioinformatics – Dillon Dugan 17 C-value paradox, nc RNA - 18 epigenetics – Madeline Jeshurin 19 RNAi – Rebeckah Ruiz 20 CRISPR – Matt Johnson 21 Phylogenetic genomics – Mariel Campbell 22 Archeological or commercial genomics -

4. SNAILAND PARASITES BIOLOGY DNA “identity, possibilities” phylogenetics CTAB/DNAzol gel electrophoresis nanodrop spec Illumina (full) genome sequencing Qubit Fluorometry Covaris fragmentation Ampure (fragment collection) Kapa DNA library preparation kit Pippin size selection QC Bioanalyzer, Qubit, qPCR Illumina run PCRrDNA/mito TA cloning, B/W screening electrophoresis Qiagen plasmid extraction Restriction digests direct sequencing M13 sequencing Sequence ID (BLAST) editing Galaxy QC Data file (MT) genome assembly Mitos, manual annotation Gene annotation Primer design, walking Phylogenetics GenBank submission

5. Specimens 36 lymnaeids 66 physids 82 planorbids 184 snails 3.2% infected snails +released cercariaefixed in 80% EtOH Shady Lakes: SNAILS AND PARASITES Physid Physella sp? 3 lymnaeid echinostome 3 physid echinostome 24 Aug 2018 Lattitude 35°12'59.15"N Longitude 106°35'54.52"W(Google Earth) echinostome

6. Specimens 16 limpets Ancylid or Acroloxid?

7. SAMPLE ASSIGNMENTS PES4 4 PE2 EP2 2 PE1 EP1 8 LE5 EL5 6 PE3 EP3 10 A2 EP3 1 PE1 EP1 5 PE3 EP3 7 LE5 EL5 3 PE2 EP21 9 A1 EP1

8. Things to be careful about with DNA Extraction Safety, gloves-lab coat Vortexing/pipetting Too much starting material? When is a good time to stop Contamination (gloves)) 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

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

10. HOMEWORK • Provide answers to questions 1-3 in the format of x ml EtOH stock + y ml water • How much volume of 100% EtOH is needed for 100 ml of a 70% EtOH dilution? • 70 ml EtOH stock + 30 ml water • 2) How much volume of 95% EtOH is needed for 95 ml of a 70% EtOH dilution? • 70 ml EtOH stock + 25 ml water • 3) How much volume of 100% EtOH is needed for 50 ml of a 80% EtOH dilution? • 40 ml EtOH stock + 10 ml water • 4) You are preparing a 0.5 M EDTA solution, the EDTA powder does not dissolve. How do you solve this problem? • EDTA only dissolves at pH ≥ 8, so increase pH • 5) You have 40nmoles of a PCR primer, how much water (in ml) do you add to make a 50 micromolar stock solution? • 0.8 ml (800 ml) • 6) You have just finished making 1 L of a 50x stock solution for a technique that you and all your 20 colleagues in the lab perform almost every day. You realize that you forgot to adjust the pH. What do you do? • MAKE THE CORRECT 50x STOCK ANEW

11. HOMEWORK • Provide answers to questions 1-3 in the format of x ml EtOH stock + y ml water • How much volume of 100% EtOH is needed for 100 ml of a 70% EtOH dilution? • 70 ml EtOH stock + 30 ml water 100 x X = 70 x 100; X =70 x 100/100 • 2) How much volume of 95% EtOH is needed for 95 ml of a 70% EtOH dilution? • 70 ml EtOH stock + 25 ml water 95 x X = 70 x 95: X = 70 x 95/95 • 3) How much volume of 100% EtOH is needed for 50 ml of a 80% EtOH dilution? • 40 ml EtOH stock + 10 ml water 100 x X =80 x 50: X =80 x 50/100 • 4) You are preparing a 0.5 M EDTA solution, the EDTA powder does not dissolve. How do you solve this problem? • EDTA only dissolves at pH ≥ 8, so increase pH • 5) You have 40nmoles of a PCR primer, how much water (in ml) do you add to make a 50 micromolar stock solution? • 0.8 ml (800 ml) • 50 micromolar= 50micromole/L = 50nmole/ml. have 40nmoles so 40/50 x 1ml= 0.8 ml • 6) You have just finished making 1 L of a 50x stock solution for a technique that you and all your 20 colleagues in the lab perform almost every day. You realize that you forgot to adjust the pH. What do you do? • MAKE THE CORRECT 50x STOCK ANEW

12. 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)

13. DNA/RNA WHERE? (shell) tissue cells nucleus/mitochondria RNA (mechanicaldisruption) DNA Also in the way, lipids, mucopolysaccharides, proteins (enzymes) Avoid all that and obtain DNA/RNA parasites/other symbionts, animal, plant, bacteria, fungi?

14. - Huelskens T, Schreiber S, Hollmann M: COI amplification success from mucus-rich marine gastropods (Gastropoda:Naticidae) depends on DNA extraction method and preserving agent. Mitteilungen der Dtsch Malakozool Gesellschaft 2011, 85:17–26. - Shioda M, Murakami-Murofushi K: Selective inhibition of DNA polymerase α by a polysaccharide purified from slime of Physarum polycephalum. Biochem Biophys Res Commun 1987, 146:61–66. - Sokolov EP: An improved method for DNA isolation from mucopolysaccharide-rich molluscan tissues. J Molluscan Stud 2000, 66:573–575. - Popa OP, Murariu D, Popa LO: Comparison of four DNA extraction methods from invasive freshwater bivalve species (Mollusca, Bivalvia) in Romanian fauna. Trav du Muséum Natl d’Histoire Nat <<Grigore Antipa>> 2007, L(October 2007):527–536.

15. 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

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

17. 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://www.mrcgene.com/dnazoffer.htm http://en.wikipedia.org/wiki/Chaotropic_agent

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

19. Alcohol precipitation Alcohol plus aqueous (UPPER) phase DNA cannot retain water in the presence of alcohol plus 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 H2O

20. 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.

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