1. Today House Keeping (schedule/additional sequencing) 15.ppt Conrad PCR; T-gradient PCR redo parasite P5 (four targets) PCR with 2 snails from Sevilleta

3. 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 Trizol TA cloning, B/W screening M13 sequencing Primer design, walking Qiagen plasmid extraction Restriction digests DNA RNA GenBank submission

4. BLAST results snail amplicon direct sequencing Groupsample16S (BLAST ID)COI (BLAST ID) 1 1F Physa fontinalisR Biomphalaria 2 2F ?R Physella acuta 3 3(-)F/R Achatina fulica, Physids 4---- 5 5F Physa fontinalisR Physella acuta 6 4F Physella acuta R Physella acuta 7 5R (-)F (++) 8 4R (Radix, Physella)F Physella acuta 9 1R Physella acuta F Physella acuta 10 2R SimuliumF (-) 1: snail, physid? 2: insect or Physella acuta? 3: snail.? 4: Radix, Physella acuta 5: physid DO NOT HAVE ALL FORWARD AND REVERSE COMPLETE GIVE ME YOUR AMPLICONS FOR ADDITIONAL SEQUENCING LABEL!!!!

5. Sequencing: Repeating 16S F+R ; CO1 S F+R Using amplicons from 1 group 1;9 2 group2 3 group3 4 group6;8 5 group5 complete sequencing, or obtain forward plus reverse

6. 15min.ppt

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. http://sev.lternet.edu/about FIELDTRIP to Sevilleta LTER, Sample collection: Sunday 13 September

9. Tm: melting temperature of primers: 50% of primers annealed to template Lower T, increased %, plus mismatches (ASPECIFIC) Higher T, reduced %, fewer/NO mismatches T (temperature) % primer bound to template 50 100 T melting Temperature gradient)

10. Compose PCR reactions. LABEL group#, reaction#: Parasite SP5 50 microliter in one tube: Group 1 and 2 for P5 COI: Aliquot: 5 x10, Group 3 and 4 for P5 16S : Aliquot: 5 x10 Group 6 and 7 for P5 18S : Aliquot: 5 x10 Group 8, 9, 10 for P5 28S: Aliquot 4 x 12.5 What are your ingredients? Also Snail SS1 50 microliter reactions: Group 1 for SS1 COI: Aliquot: 5 x10 Group 2 for SS1 16S: Aliquot: 5 x10 Group 3 for SS1 18S: Aliquot: 5 x10 Group 5 for SS1 28S: Aliquot: 5 x10 Group 6 for SS2 COI: Aliquot: 5 x10 Group 7 for SS216S: Aliquot: 5 x10 Group 8 for SS2 18S: Aliquot: 5 x10 Group 9 for SS2 28S: Aliquot: 5 x10 Group10 for SS1 18S: Aliquot: 5 x10 Group 3 and 4 for P5 16S : Aliquot: 5 x10 Group 6 and 7 for P5 18S : Aliquot: 5 x10 Group 8, 9, 10 for P5 28S: Aliquot 4 x 12.5

11. Handout 5 24  l mQ water 5  l 10X buffer 8  l MgCl 2 8  l dNTPs 1  l TaqGold (look to see it descend to the bottom) 2  l primers 2  l template