Team:Gaston Day School/cadmiumdetector

From 2014.igem.org

(Difference between revisions)
Line 318: Line 318:
                         <img src="https://static.igem.org/mediawiki/2014/thumb/6/60/Cd_construction.jpg/800px-Cd_construction.jpg" alt="Cadmium methods" style="width:100%">
                         <img src="https://static.igem.org/mediawiki/2014/thumb/6/60/Cd_construction.jpg/800px-Cd_construction.jpg" alt="Cadmium methods" style="width:100%">
                     </div>
                     </div>
-
</div>
+
</div>
                         </p>
                         </p>
<div class="col-1-10">
<div class="col-1-10">
Line 329: Line 329:
<h2 id="head">Methods</h2>
<h2 id="head">Methods</h2>
<p id="pad">
<p id="pad">
-
                          Methods
+
      <br>Starting Sensitivity Tests
-
<br>Duplicate Cadmium Experiment
+
<br>Determine which samples you will run
-
<br>1. Set up two groups of plastic tubes, with 4 mL of LB in each. The group of tubes that will hold the cadmium detector cells should also contain 4 µl of chloramphenicol.
+
<br>Find both the positive and negative control samples
-
<br>2. Add 100 µl of Cadmium detector cells to the tubes that contain chloramphenicol.
+
<br>Obtain 6 tubes for each sample
-
<br>3. Add 100 µl E. Coli to the other tubes.
+
<br>Add 4 mL of LB (and appropriate antibiotic for the sample)
-
<br>4. Add Cadmium to all tubes according to this chart:
+
<br>Add the following amounts of 1 M Cadmium Chloride to the respective tube
-
<br>5. Keep in the incubator overnight
+
<br>1 = 0 mM  = 0 ul
-
<br>6. Spin the tubes in the centrifuge for five minutes.
+
<br>2 = 1 mM  = 4.1 ul
-
<br>7. Pour off the supernatant.
+
<br>3 = 5 mM  = 20.5 ul 
-
<br>8. Resuspend in 1 mL of PBS.
+
<br>4 = 10 mM = 41 ul
 +
<br>5 = 50 mM = 205 ul
 +
<br>6 = .1 M      = 410 ul
 +
<br>Add 100ul of the appropriate cells to each tube
 +
<br>Mix well by inverting / vortexing
 +
<br>Grow overnight
 +
<br>
 +
<br>
 +
<br>Reading Sensitivity Results
 +
<br>Spin cells down for 5’ in clinical centrifuge
 +
<br>Decant supernatant  
 +
<br>Resuspend in 1 mL of 1x PBS
 +
<br>Open Logger Pro
 +
<br>Plug in SpectroVis
 +
<br>Fill one cuvette with PBS
 +
<br>Click “Experiment” “Calibrate”Spec. 1
 +
<br>DO NOT SKIP WARMUP
 +
<br>Place “0” in SpectroVis
 +
<br>Click “Finish Calibration” ”Okay”
 +
<br>Click on the rainbow mountain
 +
<br>Click “Absorbance vs. Concentration”
 +
<br>Select 600.9 nm
 +
<br>Click “Okay”
 +
<br>Take an OD 600 reading for each sample
 +
<br>CHANGE TO FLUORESCENCE
 +
<br>Experiment  Change Units  Spec. 1  Flu. 405
 +
<br>Click rainbow mountain
 +
<br>Change wavelength to 380.1 nm
 +
<br>Measure a fluorescence reading for each sample
 +
<br>If the reading is above 1:
 +
<br>Take 100 ul of the sample and place in a clean cuvette
 +
<br>Add 900 ul of 1x PBS
 +
<br>Repeat until reading is below 1
 +
<br>Record the number of dilutions
 +
<br>Calculate the “new” flu. reading
 +
<br>Divide the fluorescence reading by its OD 600 reading and record answer as Flu. / OD
 +
<br>Graph and analyze
 +
                             <div class="col-8-10">
                             <div class="col-8-10">

Revision as of 21:24, 16 October 2014

Welcome to the 2014 GDS iGEM Wiki

The surrounding areas of Duke Energy’s Buck Steam Station have unintentionally been affected with millions of tons of coal ash containing multiple toxic chemicals including Cadmium. The release of this ash has caused the water to become a health hazard with the potential to cause a wide range of symptoms: flu-like symptoms, kidney damage, fragile bones, and possibly death through prolonged exposure. To minimize the damage caused by Cadmium in water both locally and globally, our 2012 team created several heavy metal detectors, but in 2013 we decided to concentrate on the Cadmium detector. The detector responds to the presence of Cadmium with green fluorescence. In 2014, the team worked to increase the sensitivity levels of our detector. Our detector needed to be able to respond to Cadmium at low enough levels that the detection would be useful and the presence of cadmium would not already be apparent. This year, we completed the addition of the 2007 Cambridge team's sensitivity tuners to our detector. The sensitivity tuners amplify the signal received by the detector. At the end of last year, after adding the sensitivity tuners, we began to see indications of a peak at lower levels of Cadmium than we had previously thought. To define that peak, we used test points that were closer together. We discovered a peak of fluorescence and identified our detection points.

Cadmium methods


Starting Sensitivity Tests
Determine which samples you will run
Find both the positive and negative control samples
Obtain 6 tubes for each sample
Add 4 mL of LB (and appropriate antibiotic for the sample)
Add the following amounts of 1 M Cadmium Chloride to the respective tube
1 = 0 mM = 0 ul
2 = 1 mM = 4.1 ul
3 = 5 mM = 20.5 ul
4 = 10 mM = 41 ul
5 = 50 mM = 205 ul
6 = .1 M = 410 ul
Add 100ul of the appropriate cells to each tube
Mix well by inverting / vortexing
Grow overnight


Reading Sensitivity Results
Spin cells down for 5’ in clinical centrifuge
Decant supernatant
Resuspend in 1 mL of 1x PBS
Open Logger Pro
Plug in SpectroVis
Fill one cuvette with PBS
Click “Experiment” “Calibrate”Spec. 1
DO NOT SKIP WARMUP
Place “0” in SpectroVis
Click “Finish Calibration” ”Okay”
Click on the rainbow mountain
Click “Absorbance vs. Concentration”
Select 600.9 nm
Click “Okay”
Take an OD 600 reading for each sample
CHANGE TO FLUORESCENCE
Experiment  Change Units  Spec. 1  Flu. 405
Click rainbow mountain
Change wavelength to 380.1 nm
Measure a fluorescence reading for each sample
If the reading is above 1:
Take 100 ul of the sample and place in a clean cuvette
Add 900 ul of 1x PBS
Repeat until reading is below 1
Record the number of dilutions
Calculate the “new” flu. reading
Divide the fluorescence reading by its OD 600 reading and record answer as Flu. / OD
Graph and analyze

cadmium results

Retrieved from "http://2014.igem.org/Team:Gaston_Day_School/cadmiumdetector"