Other Molecular Techniques

Blotting

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

Dot Blotting

Dot Blot Scheme Isolate RNA Make dilution series Heat and dot onto membranes Make labeled probe Hybridize Wash Autoradiograph/detect

Transcription Mapping

Promoter Characterization

In vitro mutagenesis

Protein Characteristics

Protein Structure 1º- Primary –Linear sequence of amino acids/peptide bonds 2º- Secondary –Hydrogen bonds -  helix &  sheet 3º- Tertiary- organized into subunit domains –Hydrogen, ionic & disulfide bonds 4º- Quaternary- organization of multiple domains into a multi-subunit protein –Hydrogen, ionic,metallo & disulfide bonds

Physical Separation Techniques

Centrifugation

Equilibrium Centrifugation Ultra Centrifuges CsCl gradients that self-form Separates on the basis of buoyant density Molecules migrate to a specific buoyant density at equilibrium DNA migrates differentially depending on AT/GC composition RNA may pellet

Column Chromatography

Protein Electrophoresis

SDS Gel Electrophoresis

Isoelectric Focusing/ 2 Dimensional Gels

Western and Electroblotting

Peptide Mapping

Isotope Labeling

X Ray Crystallography