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

2. 15 minute powerpoint topics(G+) datetopicname 21-SepDiscovery of DNA structureJanette Mendoza 25-SepRestriction enzymesGabriela Perales 28-SepSouthern blottingCarlos GarciaG 2-OctCloningTimothy McBrideG 6-OctThe first sequenced geneConrad Greaves 13-Oct(q)PCR, specificity and sensitivity Krystal Charly 16-OctESTsIan Keller 20-OctBLAST and database searchesRyan HeimrothG 23-OctMicroarraysBianca Myers 26-OctForensicsJennifer Gutierrez 30-Oct Genome sequencing, the $1000 genome Ayesha ArefinG 2-NovNext generation sequencingLeslie Janet LopezG 6-NovBioinformaticsAmalia Parra 9-NovEpigeneticsClyde Moya 13-Novnon-coding RNAHelen Nordquist 13-NovC-value paradoxKelsey CookG 20-NovPhylogenetic genomicsJennifer Cooksey 23-Nov Genes associated with Type 1 diabetes Katie Kesler

3. http://sev.lternet.edu/about FIELDTRIP to Sevilleta LTER, Sample collection: Sunday 13 September (Sunday 20 September)

4. PARASITES AND SNAIL BIOLOGY “identity, possibilities” phylogenetics “intentions” transcriptomics PCR rDNA/mito Bioanalyzer DNA-free, direct sequencing gel electrophoresis nanodrop spec Sequence ID (BLAST) editing Phylogenetics electrophoresis RT-PCR gel CTAB/DNAzol Trizol TA cloning, B/W screening M13 sequencing Primer design, walking Qiagen plasmid extraction Restriction digests DNA RNA GenBank submission

5. Today Use your notes/handouts Make 1% agarose gels, 1 per 2 groups (i.e. 1+2; 3; 5+6; 7+8; 9+10 ) Analyze 10 microliter of each of your 4 PCR reactions. Use layer buffer with GelRed (how much?)! Lecture Results

6. MW uneven group PCR reactions 1-4 even group PCR reactions 1-4 Make 1% agarose gels, 1 gel/2 groups, (i.e. 1+2; 3; 5+6; 7+8; 9+10) Use 12 well combs Analyze 10 microliter of each of your 4 PCR reactions. Use layer buffer with GelRed (how much?) 5  l marker/lane

9. Uracil lacks this group

11. 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

12. Fig. 6-15 DNA synthesis is 5’ to 3’ and semiconservative, DNA polymerase has a proof reading capability.

13. DNA replication = DNA synthesis (template = DNA) (transfers information from generation to generation--cell and organism) Primer required Transcription = RNA synthesis (template = DNA) (transfers information from DNA to cellular metabolic machinery) Promoter not primer required

14. RNA Ribonucleic acid AUCG single stranded (5’-3’) many copies/cell variable population breakdown (hydrolysis) short messages, regulators, transporters DNA Deoxyribonucleic acid ATCG double stranded (5’-3’) one copy/cell constant stable long protein encoding, introns, regulatory sequences intergenic junk(?)

16. NOTES Protocol gel-electrophoresis? Can you independently repeat the PCR? Do you know what to expect?

17. 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 sequences or size estimates of the anticipated amplicons

18. PCR details AmpliTaq Gold, 4 mM MgCl 2 1 mM of each primer (50 picomoles) Cycling profile –10' 95C (hot start) –30x (30" 95C; 60" Tm; 60"+5" 72C) –7' 72C, –∞ 4C

19. PCR details Primers: 16S and CO1 –16S Tm 55C expected size ~600bp 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 ~700bp 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 )

20. PCR details Primers: "parasite" rDNA 18S and 28S (Olsen et al. 2003). –18S Tm 50C expected size ~1800bp 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’

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