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

GTAACGGCCCGGAGTCTGCTGGAATTCGCCCTAGGGCGAA

GTAACGGCCCGGAGTCTGCTGGAATTCGCCC TAGGGCGAACATTGCCGGGCCTCAGACGACCTTAAGCGGGAT CCCGCTT

CLOSE BUT NO QUITE

PARASITES AND SNAIL BIOLOGY

“identity, possibilities”phylogenetics

“intentions”transcriptomics

PCRrDNA/mito

BioanalyzerDNA-free,

direct sequencing

gel electrophoresisnanodrop spec

Sequence ID (BLAST)editing

Phylogenetics

electrophoresisRT-PCR

gel

CTAB

Trizol

TA cloning, B/W screening

M13 sequencing

Primer design, walking

Qiagen plasmid extraction Restriction digests

DNA

RNA

GenBank submission

G1, 28SP4 G2, 28SP4 G3, 28SP2 G5, 28SP4 G6, 18SP4

G7, 28SP4 G8, 18SP4 G9, 18SP4 G10, 28SP2

18S ~1800bp, 28S ~1400-1600 bp)

Purify extension productsDO 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.

How to see chromatograms• Sequencher

• Several other choices:• Chromas• Codoncodes• m

http://www.dnaseq.co.uk/chrom_view.html

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

NNNNNNNNNNNNNNNNNNNNNNNNNNNGNTNNNGNAAGTNNNNNNNNNNNNNGGTACNANNNNNNNNNNNNNNNNNTNTTNTCCNNANGGTAATTTANNNNNNNNNNNNCNNCNNATANNNNNCATAAAATTTTTTTAATAAAATTAGAAAAGTTTCTTTTAAGTTTTTGNNNNNNNGNNNNNCCAACAAAAAATTAGGATGTAATCTATTTTTTCTATTTAAAAAAATGTTATACACTTTTCTTTAAAAATTCTAAGGGTCTTCTCGTCTTTTTTCTAAATTACTGGTATTTTCACCAGATAAACAAATTAAAAAAACACTAATTATTATAGCTACTATTCATTACTTCTTTCATTCCAGACTACAATTAATAGCCAATTGATTATGCTACCTTAGCACAGTCAAGGTACTGCGGCCGTTAATAAAGTTACACCGGGCAGAAGATATCAATAATCTTTTAAAAAATTTTCTACTGACTATGTTTNNNNNAAACAGGCGANNN

ALSO see: http://seqcore.brcf.med.umich.edu/doc/dnaseq/interpret.html

END??

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

NNNNNNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGNNGNNNNNNNNNNNNNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNNNNNNGGGGGGGNGNGGNNNGGNNNNNNNNNNNNNNNNNA

NNNNN

Possible outcomes

Failed reactions: due to Dead chemistry (BigDye, primers)Contaminants (inhibitors) Salt/OrganicsToo much/little template (wedge)Loss of extension products (precipitation, running)

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

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

Primers ?