User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/03/31

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New Reaction/Sampling Protocol

Here's the new protocol we developed for analysis:

  1. Prepare AuNP fibers - vortex each 5 mL test tube containing fibers and combine some necessary amount into a large falcon tube.
  2. Centrifuge the falcon tube allowing the fibers to settle at the bottom and pour off the supernatant liquid.
  3. Add Tris/CaCl2 buffer and vortex again, resulting in a homogenous solution of fibers which can be equipartitioned between 20 sampling epi-tubes.
  4. Add protease to each epi tube. (1 epi-tube per each timepoint being sampled)
  5. At 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60 min stop the reaction by removing from the "incubator/shaker" and centrifuging for 30 sec.
  6. Collect the supernatant for analysis.

Notes from Protocol Prep

When trying to resuspend the fibers in solution, they were vortexed gently. The solution became colloidal (purple) in color with fibers suspended. When the supernatant was extracted out and replaced with buffer, the fibers did not reconstitute into solution as expected. The solution was sonicated; it appeared that the sonication possibly created a colloidal solution from the fibers.

Instead, the fibers will be dispersed in the individual test tubes, and concentrated buffer added. This will prevent excess transferring of fibers and dispersion of fibers into colloid.

The new protocol should be something similar to this:

  1. Suspend fibers in individual glass tubes
  2. Extract some amount of homogenous fiber solution and transfer to an epi-tube
  3. Add concentrated buffer to bring the final buffer concentration to 50 mM Tris/30 mM CaCl2
  4. Add protease solution to the mixture and begin shaking
  5. When the reaction is finished, spin the individual tube for 30 seconds and extract the supernatant for analysis

This should minimize the errors from the protocol we ran today.