today house keeping (schedule,hw) sequencing extension product precipitation (constructs) editing,...
DESCRIPTION
GTAACGGCCCGGAGTCTGCTGGAATTCGCCCTAGGGCGAATRANSCRIPT
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 ?