1. Molecular Tools

2. Recombinant DNA Restriction enzymes Vectors Ligase and other enzymes

3. DNA Cloning Restriction enzymes Plasmid vector –Selectable gene –Cloning sites –Origin of replication DNA ligase Transformation –Competent cells Selection

47. Dideoxy DNA Sequencing Dideoxy nucleotides Automated DNA sequencers

49. Polymerase Chain Reaction

50. Thermostable DNA polymerase Oligonucleotide primers NTPs + buffer PCR machine

51. Constructing, Screening & Selecting Recombinant Clones

52. Overall Scheme Make libraryPlate library Denature and transfer DNA to membranes Make labeled probe Denature & Hybridize Wash Dry and autoradiograph or detect

53. Library Construction Genomic  Library –Isolate Genomic DNA –Limited Digest with restriction enzyme –Analyze digest with gel electrophoresis –Prepare arms –Ligate digest to arms –In vitro package –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

54. cDNA Library

56. Genomic Library

62. 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

63. 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

64. Labels Radioisotopes Fluorescent Colorimetric Antibody

65. Random Priming

67. End Labeling

69. Digoxygenin Labeling

71. Hybridization Stringency

81. Clone Characterization

82. 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

83. DNA minipreps

86. PCR and Cloning

89. Protein Expressing Libraries

90. Overall Scheme Isolate RNAMake cDNA Ligate into expression Vector and transform/ transduce Plate and transfer protein to membrane Incubate with 1º and 2º antibodies Wash Detect with colorimetric or fluorescent technique