other molecular techniques. blotting probe synthesis nick translation –dna template preparation...
TRANSCRIPT
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Other Molecular Techniques
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Blotting
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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
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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
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Dot Blotting
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Dot Blot Scheme
• Isolate RNA
• Make dilution series
• Heat and dot onto membranes
• Make labeled probe
• Hybridize
• Wash
• Autoradiograph/detect
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Transcription Mapping
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Promoter Characterization
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In vitro mutagenesis
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Protein Characteristics
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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
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Physical Separation Techniques
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Centrifugation
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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
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Column Chromatography
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Protein Electrophoresis
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SDS Gel Electrophoresis
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Isoelectric Focusing/
2 Dimensional Gels
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Western and Electroblotting
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Peptide Mapping
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Isotope Labeling
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X Ray Crystallography
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