2+techiniques+in+cell+biology - Research techniques...

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Research techniques Microscopes antibody tagging - fusion proteins with GFP green fluorescent protein fractionation techniques electrophoresis, centrifugation, chromatography with epitope tags PCR and generic cloning 1
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microscopy 2 Differential interference contrast Histology slides are stained to produce contrast – without stain must use other types of scopes to get good images
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4
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a - scanning B, c, e, f - transmission d – freeze fracture 6
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Why does DNA run to the + pole? How visualize the DNA in the gel How might proteins run Options Dependent on what? How make all proteins run to bottom of GEL? How visualize complexes verses subunits? Role of SDS in PAGE 7
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Loading a submarine gel
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9 Anode Longer molecules Shorter molecules
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10 chromatography Using column of beads to separate biomolecules - sephadex affinity Often used to isolate mRNA from eukaryotic cells What unique property does mRNA have? Affinity? Put on in low salt and off in high How does this work?
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PCR 12
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PCR
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Cloning 14
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Colony carrying non- recombinant plasmid with intact lacZ gene Bacterial clone Colony carrying re- combinant plasmid with disrupted lacZ gene Recombinant bacteria Recombinant DNA plasmids Sticky ends Human DNA Fragments Human cell Gene of interest Bacterial cell amp R gene (ampicillin resistance) Bacterial plasmid Restriction site lacZ gene (lactose breakdown) 1 Isolate plasmid DNA and human DNA. 2 Cut both DNA samples with the same restriction enzyme, one that makes a single cut within the lacZ gene and many cuts within the human DNA.
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