today house keeping (schedule,hw) sequencing extension product precipitation (constructs) editing,...
DESCRIPTION
GTAACGGCCCGGAGTCTGCTGGAATTCGCCCTAGGGCGAATRANSCRIPT
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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
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GTAACGGCCCGGAGTCTGCTGGAATTCGCCCTAGGGCGAA
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GTAACGGCCCGGAGTCTGCTGGAATTCGCCC TAGGGCGAACATTGCCGGGCCTCAGACGACCTTAAGCGGGAT CCCGCTT
CLOSE BUT NO QUITE
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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
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G1, 28SP4 G2, 28SP4 G3, 28SP2 G5, 28SP4 G6, 18SP4
G7, 28SP4 G8, 18SP4 G9, 18SP4 G10, 28SP2
18S ~1800bp, 28S ~1400-1600 bp)
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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.
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How to see chromatograms• Sequencher
• Several other choices:• Chromas• Codoncodes• m
http://www.dnaseq.co.uk/chrom_view.html
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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
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NNNNNNNNNNNNNNNNNNNNNNNNNNNGNTNNNGNAAGTNNNNNNNNNNNNNGGTACNANNNNNNNNNNNNNNNNNTNTTNTCCNNANGGTAATTTANNNNNNNNNNNNCNNCNNATANNNNNCATAAAATTTTTTTAATAAAATTAGAAAAGTTTCTTTTAAGTTTTTGNNNNNNNGNNNNNCCAACAAAAAATTAGGATGTAATCTATTTTTTCTATTTAAAAAAATGTTATACACTTTTCTTTAAAAATTCTAAGGGTCTTCTCGTCTTTTTTCTAAATTACTGGTATTTTCACCAGATAAACAAATTAAAAAAACACTAATTATTATAGCTACTATTCATTACTTCTTTCATTCCAGACTACAATTAATAGCCAATTGATTATGCTACCTTAGCACAGTCAAGGTACTGCGGCCGTTAATAAAGTTACACCGGGCAGAAGATATCAATAATCTTTTAAAAAATTTTCTACTGACTATGTTTNNNNNAAACAGGCGANNN
ALSO see: http://seqcore.brcf.med.umich.edu/doc/dnaseq/interpret.html
END??
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Do not insert (a great number) of additional nucleotides, develop a "feel" for background, residue spacing is usually OK
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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)
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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
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Now put assemble sequences into contigs (sequencher),edit and BLAST.
Primers ?