Difference between revisions of "Team:UCLA/Notebook/Recombinant Expression/4 August 2015"

Latest revision as of 19:46, 7 August 2015

BCA for Concentrated Fraction

Following the manufacturer's protocol, a BCA was conducted on the concentrated sample made yesterday via a 20kD Pierce concentrator. 9 standards were used to plot the Beer's Law Calibration Curve, and ultra pure water was used as a blank. Immediately after addition of 1mL of working reagent to 100ul of concentrated Tamura sample, the solution turned a deep purple, and this qualitative observation indicated early on that the sample was concentrated to a good extent. (Judging from the 9 standards' reactions after adding working reagent, the less concentrated samples were green and the more concentrated samples were purple.) After spectroscopy, it was discovered that the concentrated Tamura sample was even more concentrated than the highest standard concentration, resulting in an absorbance reading of 2.66. This value is out of the 0.1-1.0 working absorbance range for Beer's Law. The spectrophotometer was unable to generate an estimated protein concentration in ug/mL. So, the data from the standards were plotted and an equation from the line of best fit was generated. The corresponding absorbance was then calculated from an mathematical expression, the quadratic function ( ), and found to be 2970ug/mL, which is approximately 3mg/mL, a 0.3 w/v %.

Note: The concentrated Tamura should have been diluted down until it generated an absorbance reading between 0.1 and 1.0, this would have allowed for a more accurate calculation of its concentration. This will be done next time if we run into the same problem with our purified and concentrated proteins.

Start-Up Culture for 12-mer Expression

The Silk Genetics Team streaked a plate with their sequence-verified 12-mer proteins. A colony was picked and pipetted into 10mL of autoclaved LB and 10ul of Chloramphenicol. Two start-up cultures were prepared to ensure at least one is in the optimal OD range for 1 L culturing. The tubes were put into a shaking incubator at 37 degrees Celsius and left overnight for 17 hours.

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 '''<u>BCA for Concentrated Fraction</u>'''
-Following the manufacturer's protocol, a BCA was conducted on the concentrated sample made yesterday via a 20kD Pierce concentrator. 9 standards were used to plot the Beer's Law Calibration Curve, and ultra pure water was used as a blank. Immediately after addition of 1mL of working reagent to 100ul of concentrated Tamura sample, the solution turned a deep purple, and this qualitative observation indicated early on that the sample was concentrated to a good extent. (Judging from the 9 standards' reactions after adding working reagent, the less concentrated samples were green and the more concentrated samples were purple.)  After spectroscopy, it was discovered that the concentrated Tamura sample was even more concentrated than the highest standard concentration, resulting in an absorbance reading of 2.66. This value is out of the 0.1-1.0 working absorbance range for Beer's Law.  The spectrophotometer was unable to generate an estimated protein concentration in ug/mL. So, the data from the standards were plotted and an equation from the line of best fit was generated. The corresponding absorbance was then calculated from an mathematical expression, the quadratic function ( ), and found to be 2986ug/mL, which is approximately 3mg/mL, a 0.3 w/v %.
+Following the manufacturer's protocol, a BCA was conducted on the concentrated sample made yesterday via a 20kD Pierce concentrator. 9 standards were used to plot the Beer's Law Calibration Curve, and ultra pure water was used as a blank. Immediately after addition of 1mL of working reagent to 100ul of concentrated Tamura sample, the solution turned a deep purple, and this qualitative observation indicated early on that the sample was concentrated to a good extent. (Judging from the 9 standards' reactions after adding working reagent, the less concentrated samples were green and the more concentrated samples were purple.)  After spectroscopy, it was discovered that the concentrated Tamura sample was even more concentrated than the highest standard concentration, resulting in an absorbance reading of 2.66. This value is out of the 0.1-1.0 working absorbance range for Beer's Law.  The spectrophotometer was unable to generate an estimated protein concentration in ug/mL. So, the data from the standards were plotted and an equation from the line of best fit was generated. The corresponding absorbance was then calculated from an mathematical expression, the quadratic function ( ), and found to be 2970ug/mL, which is approximately 3mg/mL, a 0.3 w/v %.
 Note: The concentrated Tamura should have been diluted down until it generated an absorbance reading between 0.1 and 1.0, this would have allowed for a more accurate calculation of its concentration. This will be done next time if we run into the same problem with our purified and concentrated proteins.