3040 Practical

• Plasmids can also function as vectors
• To get plasmids out of the cells, we subject them to alkaline lysis
• The chemistry of the column during lysis is that DNA will bind to the column under the high salt conditions
• For PCR to be successful, you need template DNA, a molar excess of primers, four "free" deoxynucleotides, and DNA polymerase
• The elements for PCR are then mixed with a buffer that contains Mg2+
• The PCR uses a series of sequential heating and cooling steps at three different temperatures in the thermal cycler
• The three steps of the PCR are denaturation, annealing, and extension
• Each PCR cycle doubles the amount of target DNA
• Ligation requires the use of ligase, which creates the final covalent bonds between the two DNA fragments' sugar phosphate backbon3es
• NcoI and NotI both leave sticky ends
• The importance of using NotI and NcoI is that the insert will be put in the correct direction and the insert will fall into the vector's appropriate reading frame
• Transformation does occur in nature and is one way bacterial antibiotic resistance can be transferred between species
• Because bacteria don't typically go around just soaking up DNA, you use competent cells, which have been specially prepared to uptake DNA
• Kanamycin is the selectable marker used in plasmid transformation
• We call the resistence gene a selectable marker since it allows us to select for only cells which took up our plasmid of interest
• With a broad range of structure and function and the very large number of proteins in a cell, a protein must often be targeted
• When a beam of light of a specific energy is able to excite a substance, often in a region known as the chromophore, the energy of the wave is partially absorbed so that the energy of the transmitted beam is less than that of the incident beam
• Absorbance is a unitless measurement
• The absorptivity coefficient is experimentally determined from a standardized plot of absorbance versus concentraion
• The extinction coefficient is most often expressed in units of inverse concentration unit times inverse pathlength
• In biochemistry, absorption and transmission are most used because these are directly related to the concentration of the substance of interest
• The wavelength maximum is the specific frequency of light where energy absorption is greatest and can be identified in an absorption spectrum as a peak
• Standard curves are often based on concentration or quantity of of a standard control and the measured absorbance of each standard
• The slope of the standard curve is represented by the extinction coefficient times the pathlength
• Destructive assays are ones in which you cannot use the sample after the assay
• BCA and Bradford assays are destructive and colorimetric
• The UV280 assay is non-destructive and non-colorimetric
• For the BCA assay, a sample of protein in solution is first mixed with a primary reagent containing a Cu(II) ion
• When exposed to alkaline and high temperature conditions, the Cu(II) ion will be reduced into a Cu(I) ion via a reaction with the peptide bonds in the protein
• In a following reaction, two molecules of BCA will bind to the Cu(I) ion resulting in an intense purple color
• The Bradford assay is based on a bathochromic or red shift in wavelength maximum of the coomassie blue G-250 dye
• The shift that occurs in the bradford assay occurs when the reagent dye binds to proteins in an acidic solution
• The G-form reagent dye binds primarily with any surface aromatic residues as opposed to the R-form, which binds primarily with any surface positively charged or basic residues
• The color shift for the Bradford assay is only linear over a small range of protein concentrations, and the color complex is not stable for extended times
• The Bradford assay is compatible with many reagents except for detergents
• The UV280 assay is quick, non-destructive, and linear but varies quantitatively with different proteins
• The variance in the UV280 assay is due to the differences of relative aromatic residue percentages
• The UV280 assay is more commonly used to monitor elution profiles
• Another common application of the UV280 method is for the rough determination of sample purity
• Following the overexpression of a target protein, subsequent techniques included cell/tissue lysis, a physical or crude separation of unwanted cellular components, intermediate protein purification, and various analytical methods