User:Beatriz Gimenez De C./Notebook/572/2015/02/10: 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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==Entry title==
==Tasks==
* Insert content here...


* Set and monitor Proteinase K Reaction
** 0.00125 g of proteinase K was dissolved in 1 mL of Tris/NaCl buffer ---> stock [proteinase K] = 43.25 μM
** Fibers centrifuge (300 RPM for 8 min) + liquid was extracted.
** Different protease concentrations added + samples were left in the incubator shaker (37C) for 2.5 hours.
** Time points collected: 0, 30 min, 60 min, 90 min, 120 min, 150 min
* Monitor through Bradford analysis samples
**  Samples run 150 min; extracting 300 μL samples every 30 minutes:
*** 0.116 mL of stock solution + 4.884 mL of Tris/NaCl buffer --> [proteinase K]<sub>final</sub> = 1 μM
*** 0.012 mL of stock solution in 4.988 mL of Tris/NaCl buffer --> [proteinase K]<sub>final</sub> = 100 nM
*** 0.001(2) mL of stock solution in 4.999 mL of Tris/NaCl buffer --> [proteinase K]<sub>final</sub> = 10 nM
*** 10 μL of stock solution in 990 μL of Tris/NaCl buffer then take 0.012 mL of dilution in 4.988 mL of Tris/NaCl buffer --> [proteinase K]<sub>final</sub> = 1 nM
* Monitor with UV-Vis
** Fibers centrifuged (300 RPM for 5 min) + liquid extracted.
** Used Ocean Optics + set to scan every 30 sec for 2 hours (at 37°C + 1000 RPM).
** 0.0075 mL of stock solution in 2.9925 mL of Tris/NaCl buffer --> [proteinase K]<sub>final</sub> = 100 nM
* Bradford Calibration Curve
** 100 μg/mL stock lysozyme + 0.023 mL of stock proteinase K (43.25 μM) --> [proteinase K] in lysozme stock = 100 nM
** 6 samples were run in order to construct a calibration curve:
*** 0.025 mL of stock lysozyme + 0.2 mL 1:4 Bradford:Tris/NaCl + 0.775 mL Tris/NaCl buffer --> [lysozyme] = 2.5 μg/mL
*** 0.05 mL of stock lysozyme + 0.2 mL 1:4 Bradford:Tris/NaCl + 0.750 mL Tris/NaCl buffer --> [lysozyme] = 5.0 μg/mL
*** 0.10 mL of stock lysozyme + 0.2 mL 1:4 Bradford:Tris/NaCl + 0.700 mL Tris/NaCl buffer --> [lysozyme] = 10.0 μg/mL
*** 0.125 mL of stock lysozyme + 0.2 mL 1:4 Bradford:Tris/NaCl + 0.775 mL Tris/NaCl buffer --> [lysozyme] = 12.5 μg/mL
*** 0.15 mL of stock lysozyme + 0.2 mL 1:4 Bradford:Tris/NaCl + 0.650 mL Tris/NaCl buffer --> [lysozyme] = 15.0 μg/mL
*** 0.20 mL of stock lysozyme + 0.2 mL 1:4 Bradford:Tris/NaCl + 0.600 mL Tris/NaCl buffer --> [lysozyme] = 20.0 μg/mL
== Results==
* Absorbance vs Time for the Proteinase K In situ reaction.
[[Image:100nM_ProtK_AbsvsTime_Chart.png|500px]]
* Kinetics at 517.7 nm.
[[Image:100nM_ProtK_Kinetics_Chart.png|500px]]
* Absorbance vs Time for the Proteinase K In situ reaction.
[[Image:Proteinase_K_100nM_Feb_10_Chart.png|500px]]
* Bradford Results
[[Image:Bradford_Calibration_Curve_Feb10-NEW.png]]


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Latest revision as of 00:40, 27 September 2017

Project name Main project page
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Tasks

  • Set and monitor Proteinase K Reaction
    • 0.00125 g of proteinase K was dissolved in 1 mL of Tris/NaCl buffer ---> stock [proteinase K] = 43.25 μM
    • Fibers centrifuge (300 RPM for 8 min) + liquid was extracted.
    • Different protease concentrations added + samples were left in the incubator shaker (37C) for 2.5 hours.
    • Time points collected: 0, 30 min, 60 min, 90 min, 120 min, 150 min
  • Monitor through Bradford analysis samples
    • Samples run 150 min; extracting 300 μL samples every 30 minutes:
      • 0.116 mL of stock solution + 4.884 mL of Tris/NaCl buffer --> [proteinase K]final = 1 μM
      • 0.012 mL of stock solution in 4.988 mL of Tris/NaCl buffer --> [proteinase K]final = 100 nM
      • 0.001(2) mL of stock solution in 4.999 mL of Tris/NaCl buffer --> [proteinase K]final = 10 nM
      • 10 μL of stock solution in 990 μL of Tris/NaCl buffer then take 0.012 mL of dilution in 4.988 mL of Tris/NaCl buffer --> [proteinase K]final = 1 nM
  • Monitor with UV-Vis
    • Fibers centrifuged (300 RPM for 5 min) + liquid extracted.
    • Used Ocean Optics + set to scan every 30 sec for 2 hours (at 37°C + 1000 RPM).
    • 0.0075 mL of stock solution in 2.9925 mL of Tris/NaCl buffer --> [proteinase K]final = 100 nM
  • Bradford Calibration Curve
    • 100 μg/mL stock lysozyme + 0.023 mL of stock proteinase K (43.25 μM) --> [proteinase K] in lysozme stock = 100 nM
    • 6 samples were run in order to construct a calibration curve:
      • 0.025 mL of stock lysozyme + 0.2 mL 1:4 Bradford:Tris/NaCl + 0.775 mL Tris/NaCl buffer --> [lysozyme] = 2.5 μg/mL
      • 0.05 mL of stock lysozyme + 0.2 mL 1:4 Bradford:Tris/NaCl + 0.750 mL Tris/NaCl buffer --> [lysozyme] = 5.0 μg/mL
      • 0.10 mL of stock lysozyme + 0.2 mL 1:4 Bradford:Tris/NaCl + 0.700 mL Tris/NaCl buffer --> [lysozyme] = 10.0 μg/mL
      • 0.125 mL of stock lysozyme + 0.2 mL 1:4 Bradford:Tris/NaCl + 0.775 mL Tris/NaCl buffer --> [lysozyme] = 12.5 μg/mL
      • 0.15 mL of stock lysozyme + 0.2 mL 1:4 Bradford:Tris/NaCl + 0.650 mL Tris/NaCl buffer --> [lysozyme] = 15.0 μg/mL
      • 0.20 mL of stock lysozyme + 0.2 mL 1:4 Bradford:Tris/NaCl + 0.600 mL Tris/NaCl buffer --> [lysozyme] = 20.0 μg/mL

Results

  • Absorbance vs Time for the Proteinase K In situ reaction.

  • Kinetics at 517.7 nm.

  • Absorbance vs Time for the Proteinase K In situ reaction.

  • Bradford Results


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