Other Molecular Techniques Blotting Probe synthesis Nick Translation

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 T 7, T 3 or Sp 6 vector Restriction cut to linearize RNA polymerization with labeled r. NTPs 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- a-helix & b-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 Cs. Cl 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