User:Nicole Bonan/Notebook/Chem 571 Lab Notebook/2015/10/07: Difference between revisions

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==Protocol==
==Protocol==
# First, we spun 4 samples of the AuNP fibers that Dr. Hartings synthesized on [[User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2015/10/02|September 2]] down and pipetted off the supernatant
# First, we spun down 4 samples of the AuNP fibers that Dr. Hartings synthesized on [[User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2015/10/02|September 2]] and pipetted off the supernatant
# We then suspended the samples in 1 mL of Tris Buffer
# We then suspended the samples in 1 mL of Tris Buffer
## The buffer was 50mM Tris and 20mM CaCl2 with pH=8
## The buffer was 50mM Tris and 20mM CaCl2 with pH=8

Revision as of 15:31, 11 October 2015

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Objective

The purpose of today's lab work was to use the Ocean Optics spectrophotometer in order to measure how the absorbance of a sample of AuNP fiber sample changed over time as it was digested by alpha-chymotrypsin.

Protocol

  1. First, we spun down 4 samples of the AuNP fibers that Dr. Hartings synthesized on September 2 and pipetted off the supernatant
  2. We then suspended the samples in 1 mL of Tris Buffer
    1. The buffer was 50mM Tris and 20mM CaCl2 with pH=8
  3. Next, we brought up one of our stock volumes of alpha-chymotrypsin to 1mL in the Tris buffer. The final concentration of the alpha-chymotrypsin was 53.517µM.
  4. We calculated the volume of each component that we needed to add to our cuvette:
    1. We needed the final volume in the cuvette to be 3mL (including the alpha-chymotrypsin), and we wanted the final concentration of alpha-chymotrypsin in the cuvette to be 1µM. Thus, we did the following calculation to determine how much alpha-chymotrypsin (designated as a) we would need to add to the cuvette (designated as c):
      [(1µmol a)/(1L c)] * [(1L)/(1000mL)] * [3mL] * [(1L a)/(53.517µmol a)]
      =5.6067x10-5L
      =56µL of the alpha-chymotrypsin would be needed
    2. We needed to add 1mL of the AuNP fiber sample. Thus, the volume of buffer that we would need to add to the cuvette in order to bring the final volume of the cuvette up to 3mL was:
      3000µL total -1000µL AuNP fiber sample - 56µL alpha-chymotrypsin
      =1944µL of Tris buffer
  5. We then added the AuNP fibers and the Tris buffer to the cuvette and began taking measurements
    1. We saved the graph and set the parameters to be 2min between scans and to stop taking absorbance measurements after 2.5 hours
    2. We clicked save, then play
  6. After the second file was created, we added the alpha-chymotrypsin to the sample.

Data


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