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Today

Today. Do you have PCR amplicons? Run gel *.ppt Background DNA and our PCRs Interpretation of PCR results What to do next?. Pour agarose gel now. Add 0.3 g agarose to 30 ml 1X TAE Microwave and pour as before (12 tooth comb. SNAIL AND PARASITES BIOLOGY. DNA. “identity, possibilities”

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Today

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  1. Today • Do you have PCR amplicons? • Run gel • *.ppt • Background DNA and our PCRs • Interpretation of PCR results • What to do next?

  2. Pour agarose gel now • Add 0.3 g agarose to 30 ml 1X TAE • Microwave and pour as before (12 tooth comb

  3. SNAILAND PARASITES BIOLOGY DNA “identity, possibilities” phylogenetics CTAB/DNAzol CTAB/DNAzol Illumina (full) genome sequencing gel electrophoresis nanodrop spec PCRrDNA/mito Qubit Fluorometry Covaris fragmentation Ampure (fragment collection) Kapa DNA library preparation kit Pippin size selection QC Bioanalyzer, Qubit, qPCR Illumina run TA cloning, B/W screening electrophoresis Qiagen plasmid extraction Restriction digests direct sequencing M13 sequencing Sequence ID (BLAST) editing Galaxy QC Data file (MT) genome assembly Mitos, manual annotation Gene annotation Primer design, walking Phylogenetics GenBank submission

  4. SNAILAND PARASITES BIOLOGY DNA “identity, possibilities” phylogenetics CTAB/DNAzol CTAB/DNAzol Illumina (full) genome sequencing gel electrophoresis nanodrop spec Next Friday Today PCRrDNA/mito Qubit Fluorometry Covaris fragmentation Ampure (fragment collection) Kapa DNA library preparation kit Pippin size selection QC Bioanalyzer, Qubit, qPCR Illumina run Monday TA cloning, B/W screening electrophoresis Qiagen plasmid extraction Restriction digests direct sequencing M13 sequencing Sequence ID (BLAST) editing Galaxy QC Data file (MT) genome assembly Mitos, manual annotation Gene annotation Primer design, walking Phylogenetics GenBank submission

  5. DNA plus RNA? P3S P3S: Physid snail Strigeid cercariae Total DNA plus RNA Illumina whole genome sequencing

  6. DNA plus RNA? P3S P3S: Physid snail Strigeid cercariae Total DNA Also Ancylid A(-) Illumina whole genome sequencing

  7. SNAILAND PARASITES BIOLOGY DNA “identity, possibilities” phylogenetics CTAB/DNAzol CTAB/DNAzol Illumina (full) genome sequencing gel electrophoresis nanodrop spec PCRrDNA/mito Qubit Fluorometry Covaris fragmentation Ampure (fragment collection) Kapa DNA library preparation kit Pippin size selection QC Bioanalyzer, Qubit, qPCR Illumina run TA cloning, B/W screening electrophoresis Qiagen plasmid extraction Restriction digests direct sequencing M13 sequencing Sequence ID (BLAST) editing Galaxy QC Data file (MT) genome assembly Mitos, manual annotation Gene annotation Primer design, walking Phylogenetics GenBank submission

  8. Outcomes determine cloning or re-PCR

  9. Today • Use your notes/handouts • Make 1% agarose gels, 10 well comb • 1 per 2 groups(i.e. 1+2; 3+4; 5+6; 7+8; 9+10m, 11+12) • *.ppt • Analyze 10 microliter of each of your 4 PCR reactions. Use layer buffer with GelRed (how much?)! • MW = “NEB 1kB” AND “l x HindIII” • Lecture • Results

  10. *.ppt Preparation Information Time References Presentation, Q&A

  11. Make 1% agarose gels,1 gel/2 groups, (i.e.1+2, 3+4, 5+6; 7+8,, 9+10, 11+12) 10 well combs Analyze 10 microliter of each of your 4 PCR reactions order 1,2,3,4, keep, store PCR reactions in freezer. Use layer buffer with GelRed (how much?) 5ml marker/lane Run 90V, constant voltage MW MW Group APCR reactions1-4 Group BPCR reactions1-4

  12. Return the remainder of your PCR products to the freezer! Do not add dye to the entire PCR sample. You will need the rest for sequencing.

  13. Anatomy of a good PCR product • Correct size • ds DNA • (Primer 1) - amplified region – (primer 2) • Checks/Controls: • Positive (did the reagents work?) • Tp1p2, Tp1-, T-p2, p1p2, T– (where T = template, p is primer) Controls

  14. DNA synthesis is 5’ to 3’

  15. Difference between hydrogen bonding (weak) of base pairs and covalent bonds (strong) of backbone extremely important Biologically information is “stored” AND can be accessed and transferred In laboratory ds DNA can be denatured and reannealed without losing information

  16. Targets • Mitochondrial rDNA, protein coding gene, Nuclear gene(s):PCR 1,2,3 : 16S, CO1, 18S or 28S (snail, more?) • Nuclear rDNA genes:PCR 4 : 18S or 28S (parasite)

  17. Targets • Mitochondrial rDNA, protein coding gene, Nuclear gene(s):PCR 1,2,3 : 16S, CO1, 18S or 28S (snail, more?) • Nuclear rDNA genes:PCR 4 : 18S or 28S (parasite)

  18. TARGET Nuclear rDNA genes Repeated rDNA gene cassette Genes occur across phylogeny

  19. Mol Biol Evol. 2002 Mar;19(3):289-301.

  20. Nolan et al., 2013

  21. NOTES Protocol gel-electrophoresis? Can you independently repeat the PCR? Do you know what to expect? (http://learn.genetics.utah.edu/content/labs/)

  22. NOTES Protocol gel-electrophoresis? YES Can you independently repeat the PCR? Do you know what to expect? NO and NO Because you were not given the needed information for the PCR reaction,the primer sequencesor size estimates of the anticipated amplicons

  23. PCR details • AmpliTaq Gold 10U/ml; 0.5U/reaction4 mM MgCl2 • 1 mM of each primer (50 picomoles) • 1x; 0.2 mM dNTPs • Cycling profile • 10' 95C (hot start) • 30x (30" 95C; 60" Tm; 60"+5" 72C) • 7' 72C, • ∞ 4C

  24. PCR details • Primers: • 16S and CO1 • 16S Tm 55C expected size ~500bp • 16SAr: 5'- CGC CTG TTT ATC AAA AAC AT -3’ • 16SBr: 5'- CCG GTC TGA ACT CAG ATC ACG T -3’(Palumbi, S. R. 1996. Nucleic acids II: the polymerase chain reaction. In: Molecular Systematics (eds. Hillis, D. M., Moritz, C. and Mable, B. K.), pp. 205–247. Sinauer & Associates Inc., Sunderland, Massachusetts.) • CO1 Tm 48C expected size ~650bp • LCO1490: 5'-GGT CAA CAA ATC ATA AAG ATA TTG G -3’ • HC02198: 5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’(Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. 1994 DNA primers for amplification of mitochondrial cytochrome C oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3, 294–299)

  25. PCR details • Primers: • "parasite" rDNA 18S and 28S (Olsen et al. 2003). • 18S Tm 50C expected size ~1800-2000bp • wormA: 5'- A/GCG AAT GGC TCA TTA AAT CAG -3’ • wormB: 5'- ACG GAA ACC TTG TTA CGA CT -3’ • 28S Tm 50C expected size ~1400bp • LSU: 5'- TAG GTC GAC CCG CTG AAY TTA AGC A -3’ • 1500R: 5'- GCT ATC CTG AGG GAA ACT TCG -3’ (Olsen et al., 2003)

  26. PCR interpretation Band? Single/multiple? Strong/weak? Correct size? WHERE TO GO NOW?

  27. Why would there be multiple bands? What problem would that create? How can we solve the problem? PCR interpretation Band? Single/multiple? Strong/weak? Correct size? WHERE TO GO NOW?

  28. Group snail (+)amplicon parasite amplicon 16S COI 18S 28S 18S 28S 1 2 P1E EP1 3 4 L1S SL1 5 6 P2E EP2 7 8 P1E EP1 9 10 L1S SL1 11 12 P2E EP2 Keep your PCR amplification products in -20C RESULTS ANTICIPATED? PROCEED (CLONING) OR REDO (PCR)? HOW?

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