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Today • Blue/White screening • Surface sterilization • Sequence editing 15 minute powerpoint topics(G+) date topic name 21-Sep Discovery of DNA structure Janette Mendoza 25-Sep 28-Sep 2-Oct Restriction enzymes Southern blotting Cloning Gabriela Perales Carlos Garcia Timothy McBride 6-Oct The first sequenced gene Conrad Greaves 13-Oct 16-Oct (q)PCR, specificity and sensitivity ESTs 20-Oct BLAST and database searches Ryan Heimroth 23-Oct Microarrays Genome sequencing , the $1000 genome Bianca Myers 26-Oct G G Krystal Charly Ian Keller Ayesha Arefin G G 30-Oct Forensics Jennifer Gutierrez 2-Nov Next generation sequencing Leslie Janet Lopez G 6-Nov 9-Nov 13-Nov 16-Nov Bioinformatics Epigenetics non-coding RNA C-value paradox Amalia Parra Clyde Moya Helen Nordquist Kelsey Cook G 20-Nov Phylogenetic genomics Jennifer Cooksey 23-Nov Genes associated with Type 1 diabetes Katie Kesler PARASITES AND SNAIL BIOLOGY DNA “identity, possibilities” phylogenetics RNA “intentions” transcriptomics CTAB Trizol gel electrophoresis nanodrop spec Bioanalyzer DNA-free, PCR rDNA/mito TA cloning, B/W screening electrophoresis direct sequencing Sequence ID (BLAST) editing Phylogenetics GenBank submission Qiagen plasmid extraction Restriction digests M13 sequencing Primer design, walking RT-PCR gel 1) Do 10 minutes 7) Spread 10 on one plate and 100 on another Monday Surface sterilize DNA SEQUENCING PCR with conserved primers, WHY? DNA PCR clean up, WHY? Amplicon (direct) sequencing, HOW? Reading extension products….. “*.ABI” Sequence analysis? sequencing 5’ 3’ A 5’ primerF + strand sense coding primerR primerF - strand antisense non-coding primerR ATCG sense GGAA A 3’ 5’ A 3’ NEED TO KNOW WHY SEQUENCE THIS? 3’ A TACG antisense CCTT 5’ sequencing forward 5’ 3’ A 5’ primerF + strand sense coding primerR primerF - strand antisense non-coding primerR ATCG sense GGAA A 3’ 5’ A 3’ Forward primer sequencing reaction 3’ 5’ ATCG A TACG 3’ - strand antisense non-coding CCTT 5’ sequencing results 5’ 3’ A primerF + strand sense coding primerR primerF - strand antisense non-coding primerR A 3’ 5’ Forward primer sequencing reaction 3’ 5’ ATCG A TACG 3’ generates sense strand ggaa - strand antisense non-coding CCTT 5’ sequencing reverse 5’ 3’ A 5’ 3’ A primerF + strand sense coding primerR primerF - strand antisense non-coding primerR ATCG sense GGAA TACG antisense CCTT A 3’ 5’ A 3’ 5’ sequencing reverse 5’ 3’ A ATCG + STRAND SENSE CODING GGAA tagc - strand antisense non-coding cctt A 3’ 5’ Reverse primer sequencing reaction 5’ ATCG SENSE 3’ 3’ A TACG antisense GGAA A CCTT 5’ CCTT 3’ 5’ sequencing results 5’ 3’ A ATCG + STRAND SENSE CODING GGAA tagc - strand antisense non-coding cctt A 3’ 5’ Reverse primer sequencing reaction 5’ ATCG tagc SENSE generates - strand 3’ GGAA A CCTT 5’ 3’ sequencing results 5’ 3’ A ATCG + STRAND SENSE CODING GGAA tagc - strand antisense non-coding cctt A 3’ 5’ Reverse primer sequencing reaction 5’ SENSE ATCG tagc generates - strand 3’ GGAA A CCTT 5’ ABI File generated 5’-3’ 5’ TTCC 3’ dnarts - setareneg cgat Sequence is reverse complement of coding sequence ! 3’ Forward primer sequencing reaction 5’ ATCG 3’ sequencing generates + strand ggaa Reverse primer sequencing reaction tagc generates - strand 5’ TTCC 3’ 5’ ATCG 3’ 3’ dnarts - setareneg sequencing generates + strand CCTT cgat ggaa reverse complement yields + (coding) sequence two confirmatory sets of sequence data 5’ Forward primer sequencing reaction 5’ ATCG 3’ sequencing generates + strand ggaa dye blobs Reverse primer sequencing reaction tagc generates - strand 3’ dye blobs CCTT 5’
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