User:Benjamin Friedel/Notebook/CHEM 471/2015/12/01

From OpenWetWare

Jump to: navigation, search
Project name Main project page
Previous entry      Next entry

Objective

Preparation of AuNP Fiber Samples for Next Day

  1. 20mL fiber samples were prepared with 1uM,100nM,10nM and 1nM alpha-chymotrypsin for overnight (24hr) incubation and data collection the following day (12/02/15)

Protease Preparation

  1. 1mL stock solution of alpha-chymotrypsin was made by adding 1mL of 50mM Tris buffer (pH=8) to dry protease for a final concentration of 53.5156µM.
    1. Through M1V1= M2V2 the correct volume of protease needed for a final incubation tube concentration of 1uM and final tube volume of 20mL was determined
      1. V1= 373.7uL
    2. The above volume was used for the 1st and 2nd replicates of the 1uM protease samples


  1. 1mL stock solution of alpha-chymotrypsin was made by adding 1mL of 50mM Tris buffer (pH=8) to dry protease for a final concentration of 57.03125µM.
    1. Through M1V1= M2V2 the correct volume of protease needed for a final incubation tube concentration of 1uM and final tube volume of 20mL was determined
      1. V1= 350.7ul
    2. The above volume was used for the 3rd replicate of the 1uM protease samples
  2. For all three 100nM protease samples M1V1=M2V2 was with final volume of 20mL to find that 35.1ul protease needed to be added to 19,964.9ul Tris buffer.
  3. For all three 10nM protease samples M1V1=M2V2 was with final volume of 20mL to find that 3.51ul protease needed to be added to 19,996.49ul Tris buffer.
  4. For all three 1nM protease samples M1V1=M2V2 was with final volume of 20mL to find that .351ul protease needed to be added to 19,999.649ul Tris buffer.
    1. This concentration is too low to accurately pipette so a 1/10 dilution of the stock protease was made to bring its concentration down to 5.703125uM and resulting in a new V1 (for the 1nM samples in 20mL of Tris buffer) of 3.51uL using the 1/10 dilution protease stock.


Incubation Tube Preparation (Samples and Blanks)

  1. Fiber samples synthesized by Dr. Hartings were used for total of 17 (1x0nM, 3x1nM, 4x10nM, 3x100nM, 3x1uM, 3x AuNP synthesis supernatant)
    1. First, AuNP fiber samples were centrifuged at 300 rpm for 10 minutes after which as much supernatant was drained as possible and collected for three of the tubes.
  2. Then, using volumes indicated above, alpha chymotrypsin and Tris buffer were added to each sample and blank tube immediately before incubation start time.
    1. Samples and supernatant tubes were incubated at 37˚ C in a water bath for 24 hours


Personal tools