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

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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

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Isoelectric Focusing/

2 Dimensional Gels

Western and Electroblotting

Peptide Mapping

Isotope Labeling

X Ray Crystallography