Objective
Preparation of AuNP Fiber Samples for Next Day
- 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
- 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.
- 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
- V1= 373.7uL
- The above volume was used for the 1st and 2nd replicates of the 1uM protease samples
- 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.
- 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
- V1= 350.7ul
- The above volume was used for the 3rd replicate of the 1uM protease samples
- 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.
- 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.
- 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.
- 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)
- Fiber samples synthesized by Dr. Hartings were used for total of 17 (1x0nM, 3x1nM, 4x10nM, 3x100nM, 3x1uM, 3x AuNP synthesis supernatant)
- 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.
- Then, using volumes indicated above, alpha chymotrypsin and Tris buffer were added to each sample and blank tube immediately before incubation start time.
- Samples and supernatant tubes were incubated at 37˚ C in a water bath for 24 hours
|
Project name
Main project page
Previous entry Next entry
