1. Today House Keeping (schedule,HW) Sequencing extension product precipitation (constructs) Editing, BLAST 30min PPT Dual activities –Editing, BLAST, direct sequencing ID –Spec plant DNA samples

3. GTAACGGCCCGGAGTCTGCTGGAATTCGCCCTAGGGCGAA

4. CATTGCCGGGCCTCAGACGACCTTAAGCGGGAT CCCGCTT CLOSE BUT NO QUITE

5. 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

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

7. Purify extension products DO NOT DISTURB THE PELLET! These are your sequencing products. 1). Set up two 1.7ml eppendorf tubes, labeled group number and target and add 100 μl 100% EtOH and 4 μl 3M NaAc. Transfer sequencing reactions from the PCR tubes to the correctly labeled tubes. Invert (mix) and spin 10’ max RPM at room temperature. 2). Discard supernate, rinse pellet with 400 μl 75% EtOH, spin 5’ max RPM at room temperature. 3). Discard supernate, take out last few drops, dry to air, give to instructor for reading of extension products.

8. How to see chromatograms Sequencher Several other choices: Chromas Codoncodes m http://www.dnaseq.co.uk/chrom_view.html

9. Chromatograms EDIT: look to see whether you can do better than the computer algorithm. Evaluate the peak pattern to Insert, delete, reassign residues First edit individual chromatograms Then align F and R sequences into a contig, allows checking, mutual confirmation

10. NNNNNNNNNNNNNNNNNNNNNNNNNNNGNTNNNGNAAGTNNNNNNNNNNNNNGGTACNANNNNNNNNNNNNNNNNNTNT TNTCCNNANGGTAATTTANNNNNNNNNNNNCNNCNNATANNNNNCATAAAATTTTTTTAATAAAATTAGAAAAGTTTCTTTTAAGTTT TTGNNNNNNNGNNNNNCCAACAAAAAATTAGGATGTAATCTATTTTTTCTATTTAAAAAAATGTTATACACTTTTCTTTAAAAATTCTAA GGGTCTTCTCGTCTTTTTTCTAAATTACTGGTATTTTCACCAGATAAACAAATTAAAAAAACACTAATTATTATAGCTACTATTCATTACTTC TTTCATTCCAGACTACAATTAATAGCCAATTGATTATGCTACCTTAGCACAGTCAAGGTACTGCGGCCGTTAATAAAGTTACACCGGGCA GAAGATATCAATAATCTTTTAAAAAATTTTCTACTGACTATGTTTNNNNNAAACAGGCGANNN ALSO see: http://seqcore.brcf.med.umich.edu/doc/dnaseq/interpret.html END??

11. Do not insert (a great number) of additional nucleotides, develop a "feel" for background, residue spacing is usually OK

12. NNNNNNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NGGNNGNNNNNNNNNNNNNNNANNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNGNNNNNNGGGGGGGNG NGGNNNGGNNNNNNNNNNNNNNNNNA NNNNN Possible outcomes Failed reactions: due to Dead chemistry (BigDye, primers) Contaminants (inhibitors) Salt/Organics Too much/little template (wedge) Loss of extension products (precipitation, running)

13. http://www.nucleics.com/DNA_sequencing_support/DNA-sequencing-failed-reaction.html ALSO see: http://seqcore.brcf.med.umich.edu/doc/dnaseq/trouble/badseq.html

14. Now put assemble sequences into contigs (sequencher), edit and BLAST. Primers ?