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Materials for Advanced methods in molecular biology
Fichas del conjunto (27)
- 1DMSO- Dimethyl SulfoxideCryoprotective agent
-Reduces risk of ice crystal formation
-Reduces freezing point of medium - 2Proteinase KSerine protease
Wide range of pH stability, optimal 6.5-9.5
Optimal temp: 65ºC
May be used with chelator - 3EDTA- Ethylenediaminetetraacetic acidChelator- binds di and trivalent metal ions
- 4IsopropanolCauses nucleic acids to precipitate because they are not soluble in alcohol.
- 5Ethanol (for DNA extraction)70-75% solution, removes salts and other water soluble impurities without resuspending the DNA/RNA
- 6RNAsesVery stable
Difficult to inactivate
No cofactors required
Need to inhibit RNAses in order to extract RNA - 7DEPC diethylpyrocarbonateProtein inhibitor
Inactivates RNAse by bonding to them covalently.
Reacts with histidine of proteins
*inactivated by heat* - 8Guanidine ThiocyanateProtein denaturant
**Inhibits RNase** - 9PhenolDenaturates 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. - 10ChloroformPhenol 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.
- 11PBS Phosphate buffered salineWater basted, osmolarity and ion concentrations which match those of the human body making it non toxic to most cells.
- 12TrypsinDigestive protease, in the case of cell growth, used to break down the ECM and detach the cells from the plate, and each other.
- 13RPMIBuffer 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.
- 14TAE bufferMixture of tris base, acetic acid, and EDTA. Typically used for elelctrophoresis. EDTA which prevents unwanted enzyme activity.
Tris is a poor buffer - 15Ethidium BromideEnters 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.
- 16SDS- Sodium dodecyl sulfateAmphiphilic 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.
- 17Triton X-100Used 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.
- 18Coomassie Brilliant BlueUsed 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.
- 19BSASerum albumin protein derived from cows. Used to create a calibration gradient using Bradford assay method.
- 20AcrylamideUsed in the form of polyacrylamide to form the gel for electrophoresis
- 21APS Ammonium persulfateUsed 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.
- 22TEMEDWorks together with APS to polymerize the acrylamide.
- 23PBSTPBS buffer with Tween 20 which is a detergent. In western blot the purpose is to reduce background staining.
- 24Dry MilkUsed as a blocking agent in immunohistochemisty to block non-specific binding sites for the antibodies.
- 25LuminolReacts, together with hydrogen peroxide and horseradish peroxidase (HPR-enzyme attached to primary antibody), to produce light.
- 26H2O2 Hydrogen peroxideOxidizes HPR, which then oxidizes the luminol, making detectable.
- 27PMSF Phenylmethylsulfonyl Fluorideserine protease inhibitor
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- Fichas sobre Materials for Advanced methods in molecular biology, creado por Sarah Winkler el 25/01/2017.
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- Sarah Winkler
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