MCAT Lab Techniques by iwantahighmcatscore.pdf

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1 IWantAHighMCATScore’s Guide to MCAT Lab Techniques
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2 Contents Gel Electrophoresis ........................................................................................................................................ 3 Native-PAGE .............................................................................................................................................. 3 SDS-PAGE .................................................................................................................................................. 3 Reducing SDS-PAGE ................................................................................................................................... 4 Isoelectric Focusing .................................................................................................................................... 4 Western Blotting (Protein) ............................................................................................................................. 4 Southern (DNA) and Northern (RNA) Blotting .................................................................................................. 5 DNA Sequencing (Sanger Dideoxynucleotide Sequencing) ................................................................................ 6 Chromatography ............................................................................................................................................ 7 Liquid Chromatography .............................................................................................................................. 7 High-Performance Liquid Chromatography (HPLC) ....................................................................................... 7 Gas Chromatography ................................................................................................................................. 7 Gel-Filtration (Size Exclusion) Chromatography ............................................................................................ 7 Ion-Exchange Chromatography ................................................................................................................... 8 Affinity Chromatography ............................................................................................................................ 8 Thin-Layer Chromatography ....................................................................................................................... 8 Distillation .................................................................................................................................................... 9 Simple Distillation ...................................................................................................................................... 9 Fractional Distillation ................................................................................................................................. 9 Vacuum Distillation .................................................................................................................................... 9 Polymerase Chain Reaction ............................................................................................................................ 9 Spectroscopy ............................................................................................................................................... 10 1 H-NMR Spectroscopy .............................................................................................................................. 10 13 C-NMR spectroscopy ............................................................................................................................. 11 IR Spectroscopy ....................................................................................................................................... 11 UV-Vis Spectroscopy ................................................................................................................................ 11 Autoradiography ......................................................................................................................................... 12 X-Ray Crystallography .................................................................................................................................. 12 Immunoprecipitation ................................................................................................................................... 12 Radioimmunoassay ...................................................................................................................................... 12 Mass Spectrometry ...................................................................................................................................... 13 Enzyme-Linked Immunosorbent Assay (ELISA) ............................................................................................... 14 Indirect ELISA .......................................................................................................................................... 14 Sandwich ELISA ........................................................................................................................................ 14 Edman Degradation ..................................................................................................................................... 15 Gram Staining .............................................................................................................................................. 15 Restriction Fragment Length Polymorphism (RFLP) ........................................................................................ 16 Salting Out and Dialysis ................................................................................................................................ 16 Reducing Sugar Tests ................................................................................................................................... 17 Tollen’s Test ............................................................................................................................................ 17 Benedict’s Test ........................................................................................................................................ 17 Fehling’s Test ........................................................................................................................................... 17 cDNA Libraries ............................................................................................................................................. 18
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3 Gel Electrophoresis Purpose: Separation of proteins, DNA, or RNA based on size and/or charge Macromolecules (proteins, DNA, or RNA) of interest are placed in the lanes of a gel. For proteins and small molecules of DNA and RNA, the gel will be polyacrylamide. For larger molecules of DNA (> 500 bp), the gel will be agarose. An electrical charge is placed across the gel. At the bottom is the positively charged anode and at the top is the negatively charged cathode . Keep in mind, since a voltage source is applied to gel electrophoresis, it follows the same principles as an electrolytic cell. Negatively charged molecules will travel toward the anode. Because of the resistance of the gel, larger molecules will have a harder time moving and thus, the molecules will be separated by size with the smallest molecules toward the bottom. The gel can then be stained for visualization, typically using Coomassie Blue dye. A lane will be loaded with a collection of molecules of a known size, called a ladder, which can used to determine the size of the molecules being ran. There are several different applications of gel electrophoresis: Native-PAGE Native-PAGE is a polyacrylamide gel electrophoresis method for proteins that occurs under non- denaturing conditions. This method will separate proteins by size while retaining their structure. SDS-PAGE SDS-PAGE is a polyacrylamide gel electrophoresis method for proteins that occurs under denaturing conditions to separate proteins by mass. Negatively-charged sodium dodecyl sulfate (SDS) is added to the solution of proteins, denatures the proteins, and binds one SDS for every two amino acids, giving all proteins the same charge-to-mass ratio. Since all proteins have the same charge-to-mass ratio, they are separated solely on mass, with the smallest proteins found toward the bottom of the gel. SDS will only interrupt non-covalent bonds, so if disulfide bridges are present in the protein, they will not be broken. This is useful when analyzing proteins with multiple subunits.
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