User:Matthew R Skorski/Notebook/471 - Exp BioChem/2016/04/05

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Objective

Today's objective is to

  • Synthesize new AuNP fibers
  • Measure the absorbance of the supernatants of the fibers synthesized on Wednesday (April 30) after incubating in Rhodamine for two hours
  • Measure the kinetics of Rhodamine absorption into the fibers (i.e., measure the UV-Vis absorption of the supernatants after the fibers incubate in Rhodamine for varying periods of time)

Protocol

Making AuNP Fibers

Before lab, I made 24 fiber samples, using the following reagents, in glass test tubes:

I added the following to each sample:

  • 892.47µL Lysozyme
  • 741.31µL Chloroauric Acid (Gold)
  • 8366.21µL DI water

I then put the samples in the oven and ran Program P1.

In lab, we used these samples for our kinetics measurements.

Making More AuNP Fiber Samples That Failed

In lab, we made an additional 32 AuNP fiber samples.

I made new chloroauric acid for use in these fibers. However, I calculated the concentration of the chloroauric acid incorrectly, which I didn't realize until Wednesday (April 6). As a result, we did not add enough chloroauric acid to our samples (by a factor of about 100), and our samples did not form fibers (or even nanoparticles; they were all clear and colorless). We discarded the failed samples.

Measuring the Kinetics of Rhodamine B In Equilibrium with AuNP Fibers

We used the AuNP fiber samples that I synthesized before lab for these measurements.

After the fibers came out of the oven, we added 71.94µM of the 1390µM stock of Rhodamine to each of 21 samples in order to bring the final concentration of Rhodamine in these samples 10 10µM. (The samples had mostly cooled at this point, but it's important to note that they were slightly warm in case this slight temperature change alters equilibrium conditions.) We covered all the samples with Parafilm, tilted them to mix the Rhodamine into solution, and then started a timer:

  • After 5 minutes, we spun 3 samples down for 2 minutes and then pipetted 3.5mL of supernatant into separate polystyrene cuvettes. We then measured the UV-Vis absorption of each sample.
  • We repeated this at 10, 15, 20, 30, 45, and 60 minutes.
    • Note: We accidentally discarded two of the 30min samples before measuring them, so we have triplicates of all the time increments except for 30min (which we have one measurement for).
  • After we measured all of these 21 samples, we measured the UV-Vis absorption of a standard by doing the following:
    • I added 71.94µM of the 1390µM stock of Rhodamine B to each of the remaining 3 samples, covered the samples in Parafilm, and tilted them to mix in the Rhodamine
    • I spun the standards down for 2 minutes
    • I pipetted 3.5mL of the supernatant off of each sample into a polystyrene cuvette and measured the UV-Vis absorption.

We saved all of the data into an Excel spreadsheet.

Measuring the UV-Vis Absorption of AuNP Fiber Supernatants After Incubating in Rhodamine B for 2 Hours

Before lab, Michael pipetted the following amounts of Rhodamine into each of the samples that were synthesized on Wednesday of last week:

After 2 hours, we spun down each sample for 2 minutes and pipetted 3.5mL of the supernatants into polystyrene cuvettes. We measured the UV-Vis absorption of each of these samples.

Results

Kinetics of Rhodamine B In Equilibrium with AuNP Fibers

See Michael's notebook

UV-Vis Absorption of AuNP Fiber Supernatants After Incubating in Rhodamine B for 2 Hours

See Michael's notebook



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