Today

• House Keeping– (field trip, ppts)

• Update on research progress– (what have we done)

• Clean up PCR reactions

• Direct sequencing

http://sev.lternet.edu/about

FIELDTRIP to Sevilleta LTER, Sample collection:

Sunday 13 September

Cook, KelseyGreaves, Conrad T.Lopez Leslie JanetMcBride, Timothy R. Mendoza, Janette Y. Parra, Amalia S. Perales, Gabriela

15 minute powerpoint topics(G+) date topic name

21-Sep Discovery of DNA structure Janette Mendoza

25-Sep Restriction enzymes Gabriela Perales28-Sep Southern blotting Carlos Garcia G2-Oct Cloning Timothy McBride G

6-Oct The first sequenced gene Conrad Greaves

13-Oct (q)PCR, specificity and sensitivity

Krystal Charly

16-Oct ESTs Ian Keller

20-Oct BLAST and database searches Ryan Heimroth G

23-Oct Microarrays Bianca Myers26-Oct Forensics Jennifer Gutierrez

30-OctGenome sequencing , the

$1000 genomeAyesha Arefin G

2-Nov Next generation sequencing Leslie Janet Lopez G

6-Nov Bioinformatics Amalia Parra9-Nov Epigenetics Clyde Moya

13-Nov non-coding RNA Helen Nordquist16-Nov C-value paradox Kelsey Cook G

20-Nov Phylogenetic genomics Jennifer Cooksey

23-NovGenes associated with Type 1

diabetesKatie Kesler

Update experiments/results

• What techniques?

• What experiments?

• What results?

• What is in our most recent reaction tubes?

PARASITES AND SNAILPARASITES AND SNAIL BIOLOGY 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

PCR results?Group S snail parasite 16S COI 18S 28S

1 1 2 2 3 3 5 5 6 4 7 5 8 4 9 1 10 2

Expect 600 700 1800 1400

+

Work schedule today• Follow protocol "spin columns"

• Each group will do a forward and a reverse direct sequencing reaction of target amplicons

• Lecture

Proceed according to handout

GroupSAMPLE TARGET/PRIMER

16S COI

1 1 F R

2 2 F R

3 3 F,R

5 5 F R

6 4 F R

7 5 R F

8 4 R F

9 1 R F

10 2 R F

http://www.qiagen.com/plasmid/anionexchangeresin.aspx

Separation of nucleic acids at neutral pH on QIAGEN Anion-Exchange Resin Elution points of different nucleic acids from QIAGEN Resin as a function of pH and NaCl concentration

SEQUENCING:POLYMERASE ACTION

ONE PRIMER

DEOXYNUCLEOTIDES VERSUS DIDEOXYNUCLEOTIDES LABEL

ALL SIZES WITH UNIQUE LABEL TO IDENTIFY THE TERMINAL NUCLEOTIDE

Sequencing PARAMETERS

PRIMER SEQUENCES

THERMAL CYCLING

BigDye step profile1’ 96C (hot top at 106C) 10” 96C15x 30” 50C or Tm 1’15" 60C 10” 96C 5x 30” 50C or Tm 1’30" 60C 10” 96C 5x 30” 50C or Tm 2’00" 60C

∞ 4C

PCR CHEMISTRY

BigDye v.3.1 (ABI) contents?[primer stock] 1.6 molartemplate 2 l SPECIFIC AMPLICONWater

16SAr_F: PU178  5' GCACCCGCTGAAYTT 3'16SBr_R: PL1642 5' CCAGCGCCATCCATTTTCA 3‘

LCO1490 F: 5'GGTCAACAAATCATAAAGATATTGG -3’HC02198 R: 5'TAAACTTCAGGGTGACCAAAAAATCA-3’

Sequencing cleanup

Cleanup of sequencing reaction extension products

WhyRemoval of unincorporated fluorescent ddNTPs (background)Removal of reaction ingredients aids downstream manipulation HowPrecipitation (remember how)Small nucleic acids (ddNTPs) do not precipitate as readily as extension products, removed with washing