User:Asya L. Tucker/Notebook/Asya 571/2015/11/04: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(Autocreate 2015/11/04 Entry for User:Asya_L._Tucker/Notebook/Asya_571)
 
No edit summary
Line 6: Line 6:
| colspan="2"|
| colspan="2"|
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
==Entry title==
==Objective==
* Insert content here...
#Measure protease Trypsin's kinetics with the fluorescence assay using 1nM of trypsin at different heating intervals.
#Create a calibration curve using fluorescence for trypsin.


==Procedure==
The general protocol detailed in Dr. Hartings' [[User:Matt_Hartings/Notebook/AU_Biomaterials_Design_Lab/2015/10/06|lab notebook]] was used. The following specific steps were performed:
*Protease Sample Prep.
#Used eppendorf tube no. 6 that weighed (1.01922 )g, and contained (0.00128)g of trypsin.
#Added 1mL of phosphate buffer.
#Final concentration: (54.93 µM
#We diluted the 54.93 uM )Trypsin sample by pipetted 0.0182 mL to 0.982 ml of phosphate buffer to make 1 uM solution of Trypsin.
#We pipetted 0.01 mL to make 0.01 uM solution of Trypsin.
*Sample Prep.
#Used 7 eppendorf tubes, each containing gold fibers.
#To each tube add:
##0.9981mL of buffer
##0.0019mL of trypsin (add this at the time of putting the tubes in the 37˚C hot water bath).
*Blank Prep.
#In on eppendorf tube add:
##0.9981mL of phosphate buffer
##0.0019mL of trypsin solution
*Additional specifications:
#Prior to prepping the samples, the eppendorf tubes containing the fibers were centrifuged for 10 mins at 300rpm.
#After heating the samples, they were all centrifuged for 1 min. at 13'200rpm. This was done only for the samples, not the blanks.
Calculations:
    V1 = [(0.1µM)(1mL)]/52.36µM = 0.0019mL, amount of trypsin solution needed
    Volume of buffer: 1mL - 0.0019mL = 0.9981mL
==Data==
[[Image:RAM_fluorescence1nmnov4.png]]


<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Revision as of 06:46, 1 December 2015

Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Objective

  1. Measure protease Trypsin's kinetics with the fluorescence assay using 1nM of trypsin at different heating intervals.
  2. Create a calibration curve using fluorescence for trypsin.

Procedure

The general protocol detailed in Dr. Hartings' lab notebook was used. The following specific steps were performed:

  • Protease Sample Prep.
  1. Used eppendorf tube no. 6 that weighed (1.01922 )g, and contained (0.00128)g of trypsin.
  2. Added 1mL of phosphate buffer.
  3. Final concentration: (54.93 µM
  4. We diluted the 54.93 uM )Trypsin sample by pipetted 0.0182 mL to 0.982 ml of phosphate buffer to make 1 uM solution of Trypsin.
  5. We pipetted 0.01 mL to make 0.01 uM solution of Trypsin.
  • Sample Prep.
  1. Used 7 eppendorf tubes, each containing gold fibers.
  2. To each tube add:
    1. 0.9981mL of buffer
    2. 0.0019mL of trypsin (add this at the time of putting the tubes in the 37˚C hot water bath).
  • Blank Prep.
  1. In on eppendorf tube add:
    1. 0.9981mL of phosphate buffer
    2. 0.0019mL of trypsin solution
  • Additional specifications:
  1. Prior to prepping the samples, the eppendorf tubes containing the fibers were centrifuged for 10 mins at 300rpm.
  2. After heating the samples, they were all centrifuged for 1 min. at 13'200rpm. This was done only for the samples, not the blanks.

Calculations: 

   V1 = [(0.1µM)(1mL)]/52.36µM = 0.0019mL, amount of trypsin solution needed
   Volume of buffer: 1mL - 0.0019mL = 0.9981mL

Data


Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Asya_L._Tucker/Notebook/Asya_571/2015/11/04&oldid=917606"