User:Andrew W Long/Notebook/CHEM-671 Research/2015/09/22: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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6 Assay samples were then created from diluted stock  
6 Assay samples were then created from diluted stock  
#*1μg/mL
 
 
'''*1μg/mL'''
*600 μL of Bio-Rad Protein Assay reagent
*600 μL of Bio-Rad Protein Assay reagent
*M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(1 μg/mL)*(3 mL) => 0.06 mL => 60 μL diluted Lysozyme stock
*M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(1 μg/mL)*(3 mL) => 0.06 mL => 60 μL diluted Lysozyme stock
*3mL total - 0.06mL stock - 0.6 mL reagent = 2.34 mL water
*3mL total - 0.06mL stock - 0.6 mL reagent = 2.34 mL water


#*3μg/mL
 
'''*3μg/mL'''
*600 μL of Bio-Rad Protein Assay reagent
*600 μL of Bio-Rad Protein Assay reagent
*M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(3 μg/mL)*(3 mL) => 0.18 mL => 180 μL diluted Lysozyme stock
*M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(3 μg/mL)*(3 mL) => 0.18 mL => 180 μL diluted Lysozyme stock
*3mL total - 0.180 mL stock - 0.6 mL reagent = 2.22 mL water
*3mL total - 0.180 mL stock - 0.6 mL reagent = 2.22 mL water


#*4μg/mL
 
'''*4μg/mL'''
*600 μL of Bio-Rad Protein Assay reagent
*600 μL of Bio-Rad Protein Assay reagent
*M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(4 μg/mL)*(3 mL) => 0.24 mL => 240 μL diluted Lysozyme stock
*M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(4 μg/mL)*(3 mL) => 0.24 mL => 240 μL diluted Lysozyme stock
*3mL total - 0.24mL stock - 0.6 mL reagent = 2.16 mL water
*3mL total - 0.24mL stock - 0.6 mL reagent = 2.16 mL water


#*5μg/mL
 
'''*5μg/mL'''
*600 μL of Bio-Rad Protein Assay reagent
*600 μL of Bio-Rad Protein Assay reagent
*M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(5 μg/mL)*(3 mL) => 0.3 mL => 300 μL diluted Lysozyme stock
*M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(5 μg/mL)*(3 mL) => 0.3 mL => 300 μL diluted Lysozyme stock
*3mL total - 0.3mL stock - 0.6 mL reagent = 2.1 mL water
*3mL total - 0.3mL stock - 0.6 mL reagent = 2.1 mL water


#*6μg/mL
 
'''*6μg/mL'''
*600 μL of Bio-Rad Protein Assay reagent
*600 μL of Bio-Rad Protein Assay reagent
*M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(6 μg/mL)*(3 mL) => 0.36 mL => 360 μL diluted Lysozyme stock
*M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(6 μg/mL)*(3 mL) => 0.36 mL => 360 μL diluted Lysozyme stock
*3mL total - 0.36mL stock - 0.6 mL reagent = 2.04 mL water
*3mL total - 0.36mL stock - 0.6 mL reagent = 2.04 mL water


#*10μg/mL
 
'''*10μg/mL'''
*600 μL of Bio-Rad Protein Assay reagent
*600 μL of Bio-Rad Protein Assay reagent
*M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(10 μg/mL)*(3 mL) => 0.6 mL => 600 μL diluted Lysozyme stock
*M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(10 μg/mL)*(3 mL) => 0.6 mL => 600 μL diluted Lysozyme stock
*3mL total - 0.6mL stock - 0.6 mL reagent = 1.8 mL water
*3mL total - 0.6mL stock - 0.6 mL reagent = 1.8 mL water


Detailed instructions can be found [[User:Matt_Hartings/Notebook/AU_Biomaterials_Design_Lab/2015/09/22|in Dr. Harting's notebook here]]. The one modification to the procedure here is that the sample volumes were 3 mL (as opposed to 1mL), and 25mg of lysozyme was added to a total of 5mL of buffer.   
Detailed instructions can be found [[User:Matt_Hartings/Notebook/AU_Biomaterials_Design_Lab/2015/09/22|in Dr. Harting's notebook here]]. The one modification to the procedure here is that the sample volumes were 3 mL (as opposed to 1mL), and 25mg of lysozyme was added to a total of 5mL of buffer.   

Latest revision as of 01:14, 27 September 2017

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Bradford Assay

Lysozyme stock solution (25.85mg) in buffer titrated to 7.505 with 1M HCl

[Tris buffer]: (25.85 mg)/( 5mL) => 5.17 mg/mL => 5,170 μg/mL

The stock was then diluted to 50ug/mL using distilled water.

M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(50 μg/mL)*(50 mL) => V1 = 0.580 μL

6 Assay samples were then created from diluted stock


*1μg/mL

  • 600 μL of Bio-Rad Protein Assay reagent
  • M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(1 μg/mL)*(3 mL) => 0.06 mL => 60 μL diluted Lysozyme stock
  • 3mL total - 0.06mL stock - 0.6 mL reagent = 2.34 mL water


*3μg/mL

  • 600 μL of Bio-Rad Protein Assay reagent
  • M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(3 μg/mL)*(3 mL) => 0.18 mL => 180 μL diluted Lysozyme stock
  • 3mL total - 0.180 mL stock - 0.6 mL reagent = 2.22 mL water


*4μg/mL

  • 600 μL of Bio-Rad Protein Assay reagent
  • M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(4 μg/mL)*(3 mL) => 0.24 mL => 240 μL diluted Lysozyme stock
  • 3mL total - 0.24mL stock - 0.6 mL reagent = 2.16 mL water


*5μg/mL

  • 600 μL of Bio-Rad Protein Assay reagent
  • M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(5 μg/mL)*(3 mL) => 0.3 mL => 300 μL diluted Lysozyme stock
  • 3mL total - 0.3mL stock - 0.6 mL reagent = 2.1 mL water


*6μg/mL

  • 600 μL of Bio-Rad Protein Assay reagent
  • M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(6 μg/mL)*(3 mL) => 0.36 mL => 360 μL diluted Lysozyme stock
  • 3mL total - 0.36mL stock - 0.6 mL reagent = 2.04 mL water


*10μg/mL

  • 600 μL of Bio-Rad Protein Assay reagent
  • M1*V1=M2*V2 => (5170 μg/mL)*(V1)=(10 μg/mL)*(3 mL) => 0.6 mL => 600 μL diluted Lysozyme stock
  • 3mL total - 0.6mL stock - 0.6 mL reagent = 1.8 mL water


Detailed instructions can be found in Dr. Harting's notebook here. The one modification to the procedure here is that the sample volumes were 3 mL (as opposed to 1mL), and 25mg of lysozyme was added to a total of 5mL of buffer.


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