CLAB 485 and CLAB 486 Learning Goals Biochemistry Laboratory I ...
CLAB 485 and CLAB 486 Learning Goals Biochemistry Laboratory I ...
CLAB 485 and CLAB 486 Learning Goals Biochemistry Laboratory I ...
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<strong>CLAB</strong> <strong>486</strong> <strong>Learning</strong> <strong>Goals</strong><br />
<strong>Biochemistry</strong> <strong>Laboratory</strong> II<br />
Nucleotides, hyperchromic effect, <strong>and</strong> DNA melting<br />
• To investigate the UV spectroscopy of nucleotide bases in RNA <strong>and</strong> DNA<br />
• To investigate the hyperchromic effect of dsDNA<br />
• To investigate the effect of heat on dsDNA<br />
• To investigate the effect of DNase on DNA structure<br />
Plasmid DNA Isolation<br />
• To investigate the effects of SDS on cell membrane rupture <strong>and</strong> cell lysis<br />
• To purify plasmid DNA from lysed cells using silica column<br />
• To determine the concentration of plasmid DNA using UV spectroscopy<br />
Restriction Analysis <strong>and</strong> Gel Electrophoresis of Plasmid DNA<br />
• To use three endonucleases to cleave plasmid DNA <strong>and</strong> control DNA<br />
• To separate digests relative to native plasmid DNA by gel electrophoresis, including pouring agarose<br />
gels.<br />
• To analyze the size of DNA <strong>and</strong> DNA fragments<br />
• To predict the size of DNA fragments <strong>and</strong> the agreement between theory <strong>and</strong> practice.<br />
Polymerase Chain Reaction (PCR) <strong>and</strong> Amplification of p-Glo Gene<br />
• To use Thermus acquatitus DNA polymerase to amplify the pGlo gene.<br />
• To purify amplified DNA<br />
• To analyze amplified DNA by agarose gel electrophoresis <strong>and</strong> endonuclease mapping.<br />
pGlo Expression<br />
• To transform a pGlo plasmid into E. coli<br />
• To investigate the effects of arabinose <strong>and</strong> ampicilin on growth of pGlo-transformed <strong>and</strong> control E. coli<br />
• To investigate the effects of arabinose <strong>and</strong> ampicilin on <strong>and</strong> expression of GFP in transformed cells.<br />
Green Fluorescent Protein (GFP) purification <strong>and</strong> polyacrylamide gel electrophoresis<br />
• To prepare overnight culture of E. coli transformed with pGlo<br />
• To lyse E. coli enriched in GFP-enriched cells.<br />
• To purify GFP by hydrophobic interaction chromatography<br />
Polyacrylamide Gel Electrophoresis <strong>and</strong> Western Blot Analysis of Green Fluorescent Protein<br />
• To determine the relative molecular mass of GRP using SDS PAGE<br />
• To identify the presence of GFP using western blot analysis of SDS PAGE gels<br />
Glycolysis –Design, Preparation, <strong>and</strong> Analysis (3 labs)<br />
• To design experiments to prove the final three steps of the glycolytic pathway.<br />
• To prepared reagents for these designed experiments<br />
• To prove experimentally the last three steps of glycolysis<br />
Allosteric control of isocitrate dehydrogenase I<br />
• To design an assay for isocitrate dehydrgenase from yeast.<br />
• To generate dose-response curves for IDH in the absence <strong>and</strong> presence of allosteric activators <strong>and</strong><br />
inhibitors<br />
• To analyze the effects of allosteric activators <strong>and</strong> inhibitors on IDH Km <strong>and</strong> Vmax kinetic constants.
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