1. Today House Keeping (schedule, PCRs 16S, COI) 30’.ppt talk Transfer of colonies Visit MBF 14.00h Complete transfer Intro editing chromatograms Edit and BLAST, ID samples

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. Snail 1; 16S; F + R Complete/ More needed

6. 30min.ppt / transfers 14.00h MBF visit

7. M13(rev) PLASMID SEQ EXPERIMENTAL PRIMER EXPERIMENTAL SEQUENCE 18S wormA: 5'- A/GCG AAT GGC TCA TTA AAT CAG -3’ wormB: 5'- ACG GAA ACC TTG TTA CGA CT -3’ 28S Tm LSU: 5'- TAG GTC GAC CCG CTG AAY TTA AGC A -3’ 1500R: 5'- GCT ATC CTG AGG GAA ACT TCG -3’ (EXPERIMENTAL PRIMER)

8. Sequencher, load sequences. Look for Vector, Primer, (remove) Remove N’s BLAST(N) the experimental sequence Groupplasmid (BLAST ID) plasmid (BLAST ID) 1 2 3 5 6 7 8 9 10 How to develop this?

9. G1, 28SP4 G2, 28SP4 G3, 28SP2 G5, 28SP4 G6, 18SP4 G7, 28SP4G8, 18SP4 G9, 18SP4 G10, 28SP2 18S ~1800bp, 28S ~1400-1600 bp)

10. 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.