You, Hamm, Adema, Natvig biology.unm/cmadema/4546_19

1. Lab Methods in Molecular BiologyBio 446-546LFALL 2019 M/F 13.00-15.50Molecular biology of snails, parasites, fungi You, Hamm, Adema, Natvig http://biology.unm.edu/cmadema/4546_19

2. Today Introductions Organization Syllabus, goals and expectations *.ppt topics, Galaxy access, Safety video Background for research project Field-trip (waiver), Introduction to pipetting Pipetting exercise/troubleshooting questions

3. Introduction Donald (Don) O Natvig Professor Fungal Genetics Coenraad (Coen) M Adema Professor Comparative Immunology/Parasitology Paris Hamm PhD Graduate Student Fungal Genetics NOT “MOLECULAR BIOLOGISTS”, USE RESEARCH TOOLS WHO ARE YOU?

5. groups • You will work in 12 groups of two • Graduate students team up with UnderGraduates • groups will be responsible for • Experimental work • Final report using experimental data • Name + picture

6. Molecular Biology • Tools for biological study • Expensive • Difficult (despite X-files and CSI) • Frustrating • Complex theory hidden in “industry-kits” • Slow • Getting bigger BIGGER BIGGER • Computer intensive BIOINFORMATICS • Great fun

7. Goals and expectations for this course Philosophy and major objective of the Course: It is easy to follow a recipe, it is harder to follow it well, and most difficult to make it work. Our major objective is to teach you to get things to work in the lab, this may not be difficult for some of you, but it may require a personality change for others. There are 5 identifiable goals in this course: (1) To introduce you to modern techniques used in molecular biology research. (2) To teach you how to obtain and apply computer- and internet-based resources for molecular biology (3) To teach you to perform research carefully so that your scientific observations stand on solid ground. (4) To troubleshoot experiments, but most of all, to persevere when nothing works. (5) To give you experience in communicating research findings to the scientific community. Ideally, this course will give you a better understanding of what a scientific career that uses molecular techniques would be like and inspire you to pursue additional studies.

8. biology.unm.edu/cmadema/4546_19

9. Syllabus ONLINE Course information such as lectures, suggested reading, handouts, and homework assignments will be posted online at: http://biology.unm.edu/cmadema/4546_18/4546.htm Textbook: NOTE there is NO mandatory book for this class. Readings will be provided through the above website in the form of protocols, scientific papers, also check out http://www.ncbi.nlm.nih.gov/sites/entrez?db=Books Recommended for those that prefer the option of a textbook for background information: Molecular Biology of the Gene (7th ed), Watson, Baker, Bell, Gann, Levine, Losick, Publised by Pearson student edition ISBN 13:978-0-321-76243-6, ISBN 10: 0-321-76243-6

10. Grading Grading (400 points undergrad/500 points for grad students) Your final grade will depend on: • midterm examination (take home) (100) • 15 minute class presentation (50) • Phylogeny exam (50) • Homework/in class troubleshooting exercises (25) • class attendance/participation (75) • final paper in journal submission manuscript format (100). • GRADUATE STUDENTS are required to give the class an additional 15 minute presentation on their (proposed) research (100). • Unexcused/excessive absences will lower your final grade. Do not make it a habit of arriving to class late. The class depends on you to be here so that we can start and finish class on time. • FYI-you will work in groups of two/three throughout the semester. You are part of a team, so don’t let your partners down. Do your share of the work, be respectful of each other’s grasp of the material, help each other whenever possible. As is common in science, you will collaborate on the writing on your final paper and turn one paper in per group. • Grade Mode: A-F, W (withdrawal) grade mode that does not impact student GPA. (Remember that the course, if dropped after relevant deadlines remains on the student record and in their tuition account)  Like before, this grade mode does require a Last Date of Attendance (LDA).  Please see Fastinfo 3489. https://unm.custhelp.com/app/answers/detail/a_id/3489/kw/grade%20mode/session/L3RpbWUvMTM0NDg4MDA2Mi9zaWQvS21FTS1CM2w%3D

11. Lab Notebook: • You will keep a lab notebook for this class. This should be a running commentary with your ideas and notes to yourself, together with all of the raw data and any calculations you make from the raw data. Information about the equipment you use should be listed as should information about the chemicals you use. It should be possible for someone generally familiar with your area of study to pick up your notebook and repeat or continue your experiments. • All of the experiments will require making calculations or summarizing your results in a notebook. This should be done during the scheduled lab period for two reasons. • First, it is usually easier to do calculations when the data is fresh in your mind and you are more accurate in recalling details for your summaries when you write them immediately. • Second, the instructor will be available during the lab period and may not be available later. One of the criteria on which you will be graded is how current you notebook is. Lab notebooks will be checked by us periodically. • You will not be evaluated on the basis of the laboratory notebook you will keep. BUT You should record everything of interest to you in the lab in this notebook. It is not necessary to reproduce methods spelled out in the handouts, but everything else should be in your notebook. You should be able to repeat your work exactly based on the your notebook. Not everything has to be polished, for example, if you want to do a small calculation, do it in your notebook, not on a paper towel (formerly the record keeping choice of most scientists). Within the last few years society has given clear signals that it expects scientists to keep reasonable records and we must all learn to do this. All written communication in the lab will be in this notebook, including feedback from the instructors. Do not write your raw data on paper towels intending to transfer it to the notebook later. If you want to have a neat record of your results and feel you cannot do this as the experiment progresses, take the raw data on one page, and then copy it to a later page in the notebook.

12. RecommendationsNotebook keeping Lab Notebook: General Guidelines: • 1. Use a note ook of decent size with numbered pages (hand numbering is okay). • 2. Make all entries in ink, beginning with your name and some identification about the notebook. • 3. Leave 5 pages or more in the front for a table of contents. For each experiment or set of closely related experiments, make an entry in the table of contents followed by the date of the experimental work, and the page number where the research is described in detail. • 4. Each experiment should list the • Title and date • One or two sentences about the experiment • Methods (including calculations and materials) • Summary of the results (generally with some sort of graph or diagram) • Short conclusion stating important findings and/or problems encountered. • 5. Changes on previous pages, when needed, should be made by crossing out the old information with one line and putting the new information nearby. Date any changes made to your notebook. • 6. Raw data such as photos of your gels can be cut out and taped into the notebook. When particularly voluminous raw data is involved, it is best to store this separately, but with a good cross reference system so that the data and the description of the data can be put together later. • 7. Rough graphs can be drawn directly in the notebook; more formal graphs should be drawn on appropriate graph paper or with a graphing program, and taped into the notebook.

14. TOPICS.ppt 1 DNA discovery 2 Restriction Enzymes 3 Southern Blotting 4 Cloning 5 First sequencing methods/gene 6 BAC Libraries 7 PCR 8 ESTs 9 BLAST/GenBank 10 microarrays 11 qPCR 12 Mitogenomics 13 Genome sequencing 14 Second generation (NGS) 15 Long read NGS 16 Forensics 17 Bioinformatics 18 C-value paradox, nc RNA 19 Epigenetics 20 RNAi 21 CRISPR 22 Phylogenetic genomics 23 Archeological genomics 24 Commercial, personal genome sequencing Pick 1st and 2nd choice, ORDER (DATE) is FIXED E-mail psh102@unm.edu, by Friday 31 Aug, first come, first served Assigned thereafter

15. Confirm your UNM e-mail address badgettj@unm.edu gbala@unm.edu sbeuchelt@unm.edu dbowman11@unm.edu bcapshaw@unm.edu RColica@salud.unm.edu stevendo@unm.edu jgarcia929@unm.edu shindi@unm.edu jehles@unm.edu mlope24@unm.edu a0012252301@unm.edu cymartin@unm.edu kmcquirk@unm.edu kynimo3@unm.edu lepaez@unm.edu spatel8@unm.edu apittis@unm.edu tshelp@unm.edu cmtucker5@unm.edu savega@unm.edu evenhaus@unm.edu westc@unm.edu yogip@unm.edu

16. Safety videohttps://www.youtube.com/watch?v=sTzW6XENAXQ

17. Working in the lab • Laboratory Rules and Guidelines: • Be safe working with dangerous equipment and chemicals, • Ask the instructors when you have questions. • Be respectful of slower working groups, they often get the better results • Laboratory etiquette (for your safety): • No food (this includes chewing gum) or drink is allowed in the lab. • No cell phones, music players and other electronic devices, No recreational browsing • Books (other than your notebook and lab manual), backpacks, clothing, and personal belongings should not be kept on lab benches. • Lab coats are recommended when conducting experiments. Wear gloves /protective eyewear whenever working with chemicals or solutions. No open toe shoes in the lab. • Handle all equipment carefully and don’t goof around in the lab. • Wash and rinse all glassware when you finish using it. • Return individual items to your drawer at the end of each lab. • Notify the instructor if any spills occur or if equipment is damaged, and assist as instructed for the clean-up. • Notify the instructor if anything is spilled on your lab notebook or manual. It is important not to take contaminated material out of the lab. • Waste disposal: • Collected in class/ in biohazard bags (tips, tubes, plates, gels) • Sharps disposal: • Glass and sharp objects should be disposed of in special containers found around the lab. Never, under any circumstances, put sharp things in the regular trash. This presents a very real hazard for janitorial staff.

18. SCHISTOSOMIASIS:A GLOBAL PROBLEM • >207 million humans infected • Annual mortality 280,000 • Morbidity 120 million humans • 600 million more humans at risk • Disease impact of schistosomiasis second only to malaria (WHO) • Effective chemotherapy, praziquantel currently used in mass treatment program targeting morbidity by the Schistosomiasis Control Initiative. • Concern for longevity of parasites, parasite resistance, un-sustainability of ongoing mass treatment efforts, absence of new drugs and vaccine. • Continued study of all aspects of schistosomiasis remains valid,

19. THE LIFE CYCLE OF Schistosomamansoni MALE adult worms develop in mesenteric veins cercariae penetrate human skin cercariae released into water FEMALE spined eggs passed in the feces cercariae produced in the snail digestive gland miracidium hatches from egg in freshwater, penetrates snail asexual reproduction occurs in freshwater snails of the genus Biomphalaria

20. http://www.dpd.cdc.gov/DPDx/Default.htm

21. http://www.biologie.uni-erlangen.de/parasit/contents/research/echino.htmlhttp://www.biologie.uni-erlangen.de/parasit/contents/research/echino.html Alaria americanum Apophallus muehlingi cal.vet.upenn.edu/merial/Trems/fascio_c.htm http://parasitology.informatik.uni-wuerzburg.de/login/n/h/0103.html greenmuseum.org/c/vban/trematode.php

22. Shady lakes facebook

23. Fieldtrip waiver Code of conduct UNM Waiver List of all names Use your own transport, Share rides as possible Meet on site at 13.30h Collect snails (and parasites?) Depart no later than 15.00h Snail info online in “class 2”

24. SNAILAND PARASITES BIOLOGY DNA “identity, possibilities” phylogenetics CTAB/DNAzol gel electrophoresis nanodrop spec Illumina (full) genome sequencing Qubit Fluorometry Covaris fragmentation 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

25. Pipettors, volume ranges P20: 2(1)-20 µl = .002-.020 ml (use “yellow” tips) 1 0 = 10.2 µl 2 P200: 20-200 µl = .020-.200 ml (use “yellow" tips) 1 0 = 102 µl 2 P1000: 100-1000 µl = .100-1.0 ml (“blue tips”) 1 0 = 1020 µl (1.02 ml: DO NOT exceed volume range) 2

26. Pipetting demonstration • Price of pipettor • Volume • Range, adjust volume • Tips correct size, double jam, twist (hold box!) PIPET: • Load: “first stop”, then put in liquid, pre-wet, slow release of plunger, view volume • Dispense: “second stop”, look for drop or bubble • Mix: down, up and down to first stop • Move tip out of liquid • Eject tip

27. Pipetting exercise • LEARN HOW TO PIPETTE • Tips • Hold the pipettor vertically • Use a smooth up and down motion • Pre-wetting your tip improves accuracy