1. Today House Keeping (talks, sequencing) Follow-up on insert check Sequencing Plant DNA extractions 15 minute PPT Sequencher plus BLAST

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

3. G1, 28SP4 G2, 28SP4 G3, 28SP2 G5, 28SP4 G6, 18SP4 G7, 28SP4G8, 18SP4 G9, 18SP4 G10, 28SP2

4. Work schedule today Each group handout. “Purelink Quick plasmid extraction" for 4 samples (1.5 ml bacterial culture). EcoRI digest on each sample for insert check on gel. Talk Sequencher https://www.thermofisher.com/us/en/home/r eferences/protocols/nucleic-acid-purification- and-analysis/dna-protocol/pre-cast-gels-for- medium-high-throughput-nucleic-acid- analysis.html

5. Some questions: Did you obtain transformants? What is the composition of plasmids in W and B colonies? How do you recover plasmids? What likely happens in the Purelink Quick plasmid extraction protocol? How can you check for the size of the insert?

6. Do you have the desired insert? How to check Presence, Size of insert

8. Gelelectrophoresis Cast gel, load samples, run, take photo…… How long is that going to take?

9. Gelelectrophoresis

10. What to expect? Insert size: Plasmid size: Pattern on gel: What primers to use? Or digest? http://bitesizebio.com/13524/how-to-identify-supercoils-nicks-and-circles-in-plasmid-preps/

11. http://arbl.cvmbs.colosta te.edu/hbookshttp://arbl.cvmbs.colosta te.edu/hbooks/genetics/b iotech/gels/agardna.html

12. Do you have the desired insert? How to check Presence, Size of insert Plasmid(linear) insert 1 2 3 4 EcoRI digest of constructs