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Description
Flashcards by Sarah Winkler, updated more than 1 year ago
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Created by Sarah Winkler
almost 8 years ago
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| Question | Answer |
| DMSO- Dimethyl Sulfoxide | Cryoprotective agent -Reduces risk of ice crystal formation -Reduces freezing point of medium |
| Proteinase K | Serine protease Wide range of pH stability, optimal 6.5-9.5 Optimal temp: 65ºC May be used with chelator |
| EDTA- Ethylenediaminetetraacetic acid | Chelator- binds di and trivalent metal ions |
| Isopropanol | Causes nucleic acids to precipitate because they are not soluble in alcohol. |
| Ethanol (for DNA extraction) | 70-75% solution, removes salts and other water soluble impurities without resuspending the DNA/RNA |
| RNAses | Very stable Difficult to inactivate No cofactors required Need to inhibit RNAses in order to extract RNA |
| DEPC diethylpyrocarbonate | Protein inhibitor Inactivates RNAse by bonding to them covalently. Reacts with histidine of proteins *inactivated by heat* |
| Guanidine Thiocyanate | Protein denaturant **Inhibits RNase** |
| Phenol | Denaturates proteins- effective in purifying DNA and RNA from proteins. When mixed with aqueous solution of DNA/RNA and proteins, it causes the protein to denaturate and precipitate into the phenol, the higher density of phenol compared to water causes it to sink in centrifugation along with the proteins, and the DNA/RNA in the water to rise to the top for easy extraction. |
| Chloroform | Phenol retains 10-15% water during the precipitation process, mixed with chloroform it reduces this effect. Is miscible with phenol and has a higher density, resulting in a more effective separation from the aqueous phase. |
| PBS Phosphate buffered saline | Water basted, osmolarity and ion concentrations which match those of the human body making it non toxic to most cells. |
| Trypsin | Digestive protease, in the case of cell growth, used to break down the ECM and detach the cells from the plate, and each other. |
| RPMI | Buffer rich in phosphate, for use in 5% CO2 atmosphere. pH 8. Contains protease inhibitor which stops the activity of the trypsin, which could damage the cells if left to work. Used for cell medium growth because of incubation time in 5% CO2 incubator. |
| TAE buffer | Mixture of tris base, acetic acid, and EDTA. Typically used for elelctrophoresis. EDTA which prevents unwanted enzyme activity. Tris is a poor buffer <pH 7. Cation buffer. |
| Ethidium Bromide | Enters the DNA between the double strands (can be used for RNA because RNA has a tendency to form folded structures causing it to resemble a double stranded structure). Emits fluorescence allowing for detection in a UV camera. |
| SDS- Sodium dodecyl sulfate | Amphiphilic detergent. Protein denaturation by disrupting non covalent bonds. Gives protein a negative charge which in turn creates electrostatic repulsion causing the protein to form a rod-like shape. Eliminating the proteins native shape as a factor for gel separation. |
| Triton X-100 | Used for cell lysis by making the membrane more permeable. Does not denaturate proteins, making it ideal for protein extraction where the shape of the protein is relevant. |
| Coomassie Brilliant Blue | Used in the Bradford method. Original reddish color. Transfers electrons to protein, changing shape of protein and exposing internal positive charge, unstable protein then binds to dye which is unstabalized by the transfer of electrons, after they bind each other the dye turns blue. |
| BSA | Serum albumin protein derived from cows. Used to create a calibration gradient using Bradford assay method. |
| Acrylamide | Used in the form of polyacrylamide to form the gel for electrophoresis |
| APS Ammonium persulfate | Used with TEMED to catalyze the polymerization of acrylamide to form a gel. Powerful oxidizing agent. Forms oxygen free radicals in aqueous solution causing the acrylamide to polymerize. Dangerous to inhale, must be used in the chemical hood. |
| TEMED | Works together with APS to polymerize the acrylamide. |
| PBST | PBS buffer with Tween 20 which is a detergent. In western blot the purpose is to reduce background staining. |
| Dry Milk | Used as a blocking agent in immunohistochemisty to block non-specific binding sites for the antibodies. |
| Luminol | Reacts, together with hydrogen peroxide and horseradish peroxidase (HPR-enzyme attached to primary antibody), to produce light. |
| H2O2 Hydrogen peroxide | Oxidizes HPR, which then oxidizes the luminol, making detectable. |
| PMSF Phenylmethylsulfonyl Fluoride | serine protease inhibitor |
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