Difference between revisions of "Team:RHIT/Experiments"

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<br> <p> Please see our <a href = "https://2018.igem.org/Team:RHIT/Notebook"> Protocols </a> for our experiments here!
 
<br> <p> Please see our <a href = "https://2018.igem.org/Team:RHIT/Notebook"> Protocols </a> for our experiments here!
 +
 +
<h1> Glycolate Oxidase Assay </h1>
 +
<p> Time permitting, we were planning to characterize and test our glycolate oxidase gene using the following assay procedure: </p>
 +
<ul><li>Mix 0.94mL of mono-potassium phosphate with 0.6mL of di-potassium phosphate with 90 mL DI H2O to generate 100mL stock solution of potassium phosphate buffer at 8pH. </li>
 +
<li>Follow Freeze/Thaw lyse protocol instructions. Note best results happen when using fresh lysate. If not possible follow step and aliquote out smaller volumes to freeze.
 +
<img src = "https://static.igem.org/mediawiki/2018/6/69/T--RHIT--assay1.jpg"><img src = "https://static.igem.org/mediawiki/2018/d/da/T--RHIT--assay2.jpg">
 +
</li>
 +
<li>Create Enzyme Assay master mixture 10mL:<br>
 +
666.7 micromoles of potassium phosphate<br>
 +
0.67 micromoles of DCIP <br>
 +
0.33 mL of 1% PMS <br>
 +
0.033 - 0.33 mL of cell extract <br>
 +
= final volume should be ~10 mL<br>
 +
Aliquote out 1 mL volumes for enzyme assay
 +
</li>
 +
<li>Create Positive Control 1 mL:<br>
 +
0.067 micromoles of DCIP <br>
 +
0.033 mL of 1% PMS <br>
 +
0.0033 - 0.033 mL of cell extract<br>
 +
Top off with potassium phosphate buffer for 1 mL volume<br>
 +
</li>
 +
<li>Create Negative Control 1 mL:<br>
 +
0.067 micromoles of DCIP <br>
 +
0.033 mL of 1% PMS <br>
 +
Top off with potassium phosphate buffer for 1 mL volume
 +
</li>
 +
<li>Make up a solution of potassium glycolate by mixing Add 10 micromoles of glycolic acid (MW: 76.05 g/mol) to enough buffer to neutralize it to glycolate (this should be enough for three 1 mL cuvette)</li>
 +
<li>Measure 600 nm time-lapse spectrophotometer for positive control over 5 mins (Absorbance start at or be under 1) </li>
 +
<li>Add 3.33 micromoles of potassium glycolate to the negative control mixture </li>
 +
<li>Measure 600 nm time-lapse spectrophotometer for negative control over 5 mins (Absorbance start at or be under 1) </li>
 +
<li>Add 3.33 micromoles of potassium glycolate to the enzyme mixture</li>
 +
<li>Measure a decrease in extinction at 600 nm time-lapse spectrophotometer over 5 mins (Absorbance start at or be under 1)</li></ul>
 +
Analysis: A decrease in extinction of 0.01 corresponds to oxidation of 1.8 nmoles of glycolate<br>
 +
 +
Adapted from J.M. Lord 1971
 
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Revision as of 23:57, 15 October 2018




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Description
Design
Experiments
Notebook
Results
Achievements
InterLab
Attributions

Experiments

Unfortunately, we were not able to do as many experiments or testing as we would have liked due to time constraints. The work we were able to do and the protocols we followed are described on our notebook page.


Please see our Protocols for our experiments here!

Glycolate Oxidase Assay

Time permitting, we were planning to characterize and test our glycolate oxidase gene using the following assay procedure:

  • Mix 0.94mL of mono-potassium phosphate with 0.6mL of di-potassium phosphate with 90 mL DI H2O to generate 100mL stock solution of potassium phosphate buffer at 8pH.
  • Follow Freeze/Thaw lyse protocol instructions. Note best results happen when using fresh lysate. If not possible follow step and aliquote out smaller volumes to freeze.
  • Create Enzyme Assay master mixture 10mL:
    666.7 micromoles of potassium phosphate
    0.67 micromoles of DCIP
    0.33 mL of 1% PMS
    0.033 - 0.33 mL of cell extract
    = final volume should be ~10 mL
    Aliquote out 1 mL volumes for enzyme assay
  • Create Positive Control 1 mL:
    0.067 micromoles of DCIP
    0.033 mL of 1% PMS
    0.0033 - 0.033 mL of cell extract
    Top off with potassium phosphate buffer for 1 mL volume
  • Create Negative Control 1 mL:
    0.067 micromoles of DCIP
    0.033 mL of 1% PMS
    Top off with potassium phosphate buffer for 1 mL volume
  • Make up a solution of potassium glycolate by mixing Add 10 micromoles of glycolic acid (MW: 76.05 g/mol) to enough buffer to neutralize it to glycolate (this should be enough for three 1 mL cuvette)
  • Measure 600 nm time-lapse spectrophotometer for positive control over 5 mins (Absorbance start at or be under 1)
  • Add 3.33 micromoles of potassium glycolate to the negative control mixture
  • Measure 600 nm time-lapse spectrophotometer for negative control over 5 mins (Absorbance start at or be under 1)
  • Add 3.33 micromoles of potassium glycolate to the enzyme mixture
  • Measure a decrease in extinction at 600 nm time-lapse spectrophotometer over 5 mins (Absorbance start at or be under 1)
Analysis: A decrease in extinction of 0.01 corresponds to oxidation of 1.8 nmoles of glycolate
Adapted from J.M. Lord 1971