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Molecular Tools Recombinant DNA • Restriction enzymes • Vectors • Ligase and other enzymes DNA Cloning • Restriction enzymes • Plasmid vector – Selectable gene – Cloning sites – Origin of replication • DNA ligase • Transformation – Competent cells • Selection QuickTime™ and a Sorenson Video decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Dideoxy DNA Sequencing • Dideoxy nucleotides • Automated DNA sequencers QuickTime™ and a Animation decompressor are needed to see this picture. Polymerase Chain Reaction Polymerase Chain Reaction • Thermostable DNA polymerase • Oligonucleotide primers • NTPs + buffer • PCR machine QuickTime™ and a Sorenson Video decompressor are needed to see this picture. Constructing, Screening & Selecting Recombinant Clones Overall Scheme Make library Plate library Denature and transfer DNA to membranes Make labeled probe Dry and autoradiograph or detect Wash Denature & Hybridize Library Construction • Genomic l Library – Isolate Genomic DNA – Limited Digest with restriction enzyme – Analyze digest with gel electrophoresis – Prepare l arms – Ligate digest to arms – In vitro package l – Infect and plate • cDNA/expression – Isolate RNA – Poly A isolation via oligo dT column – Reverse transcribe mRNA – Digest or hydrolyze mRNA – 2nd strand synthesis – Add linkers – Prepare vector with digest and phosphatase – Ligate, transform and plate cDNA Library Genomic Library Probe synthesis • Nick Translation – – – – DNA template preparation Nick template with DNase I Fill in gaps with DNA polymerase and labeled nucleotides Denature and hybridize • Random Priming – – – – DNA template preparation Anneal with random hexamers Primer extend with DNA polymerase and labeled nucleotides Denature and hybridize Probe synthesis • End labeling – Prepare template – End label with labeled ATP and polynucleotide kinase – Denature and hybridize • RNA probes – – – – Clone template into a T7, T3 or Sp6 vector Restriction cut to linearize RNA polymerization with labeled rNTPs Denature and hybridize Labels • • • • Radioisotopes Fluorescent Colorimetric Antibody Random Priming End Labeling Digoxygenin Labeling Hybridization Stringency Clone Characterization Characterization Scheme • • • • Grow up the isolated clones Make DNA from those clones Restriction digest characterization Blot to membrane and hybridize with labeled cDNA to map transcript • Auto radiograph • Subclone and DNA sequence DNA minipreps QuickTime™ and a None decompressor are needed to see this picture. QuickTime™ and a TIFF (U ncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (U ncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. PCR and Cloning Protein Expressing Libraries Overall Scheme Isolate RNA Make cDNA Ligate into expression Vector and transform/ transduce Plate and transfer protein to membrane Detect with colorimetric or fluorescent technique Wash Incubate with 1º and 2º antibodies