Difference between revisions of "Team:Exeter/Interlab"

Revision as of 16:01, 19 October 2016 (view source)

Revision as of 19:28, 19 October 2016 (view source)

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The protocol was improved by using a latin rectangle arrangement in another 96 well plate. A lab robot was used to pipette out the corresponding cultures to their wells according to our preset programme linked to a descrambling spreadsheet. The Latin rectangle is designed so that no sample gets pipetted more than once in any row or column. The order in which the samples get arranged was worked out using the random number function of Microsoft Excel. Sterile LB was loaded into peripheral wells to lessen edge effects which can skew results due to high evaporation at the periphery. These adaptations aim to provide more accurate data by reducing plate effects, samples are spread randomly around the plate so as to minimise differences in results due to irregularities in plate temperature.

−

The measurements were taken using the same calibration settings as before on the Tecan plate reader every hour for 6 hours. The plate was placed into mini shaking incubator at 37°C between plate readings. A scratch was noticed on the plate lid and was replaced. This lid was cold compared to the incubated plate and so condensation formed this could have affected the results. Placed in incubator briefly to warm up. The results were ~~complied~~ into graphs using the iGEM Interlab excel document (Fig. 3 and Fig. 4).

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+

+

Fig.3

+

+

+

</div>

+

The measurements were taken using the same calibration settings as before on the Tecan plate reader every hour for 6 hours. The plate was placed into mini shaking incubator at 37°C between plate readings. A scratch was noticed on the plate lid and was replaced. This lid was cold compared to the incubated plate and so condensation formed this could have affected the results. Placed in incubator briefly to warm up. The results were compiled into graphs using the iGEM Interlab excel document (Fig. 4 and Fig. 5).

<img src="https://static.igem.org/mediawiki/2016/1/1b/T--Exeter--interlabgraphforpart1egl.png"

style="max-width:100%;margin:auto;display:block;">

−

Fig. 3 Fluoresce/Abs 600 measurements using iGEM Interlab layout

+

Fig. 4 Fluoresce/Abs 600 measurements using iGEM Interlab layout

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<img src="https://static.igem.org/mediawiki/2016/a/a6/T--Exeter--latinrectangleinterlabgraphforpart1egl.png"

style="max-width:100%;margin:auto;display:block;">

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Fig. 4 Fluoresce/Abs 600 measurements using Latin Rectangle layout

+

Fig. 5 Fluoresce/Abs 600 measurements using Latin Rectangle layout

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An initial OD600 reading was taken using a cuvette reader. Inoculation calculations were then carried out and the cultures of the constructs were grown in new 15ml falcon tubes with 5ml of fresh LB and 5μl of chloramphenicol from a starting OD of 0.02 for 6 hours in a 37°C incubator.

−

The measurements for fluorescence were taken using a BD FACSAria II and the calibration as well as measurement protocols were followed according to the 2016 InterLab Worksheet for Flow Cytometry that is available on the iGEM website. 10,000 events were acquired from calibration beads and from each biological sample. Samples were excited using a 488 nm laser, the filter used was 530/30. Resulting graphs from the FACS measurements are shown below (Fig. 5 - Fig. 10)

+

The measurements for fluorescence were taken using a BD FACSAria II and the calibration as well as measurement protocols were followed according to the 2016 InterLab Worksheet for Flow Cytometry that is available on the iGEM website. 10,000 events were acquired from calibration beads and from each biological sample. Samples were excited using a 488 nm laser, the filter used was 530/30. Resulting graphs from the FACS measurements are shown below (Fig. 6 - Fig. 11)

Our results along with the completed worksheet were submitted on the 1/9. No problems were encountered using the protocol or FACS machine.

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<img src="https://static.igem.org/mediawiki/2016/7/7b/T--Exeter--facs1.jpg"

style="max-width:100%;margin:auto;display:block;">

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Fig. 5

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Fig. 6

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<img src="https://static.igem.org/mediawiki/2016/2/23/T--Exeter--facs2.jpg"

style="max-width:100%;margin:auto;display:block;">

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Fig. 6

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Fig. 7

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<img src="https://static.igem.org/mediawiki/2016/5/53/T--Exeter--facsp1.jpg"

style="max-width:100%;margin:auto;display:block;">

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Fig. 7

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Fig. 8

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<img src="https://static.igem.org/mediawiki/2016/2/23/T--Exeter--facsp2.jpg"

style="max-width:100%;margin:auto;display:block;">

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Fig. 8

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Fig. 9

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<img src="https://static.igem.org/mediawiki/2016/6/6c/T--Exeter--facsp3.jpg"

style="max-width:100%;margin:auto;display:block;">

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Fig. 9

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Fig. 10

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<img src="https://static.igem.org/mediawiki/2016/f/f0/T--Exeter--facs0.jpg"

style="max-width:100%;margin:auto;display:block;">

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Fig. 10

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Fig. 11

Contruct	Starting OD
P1	0.701
P2	0.614
P3	0.615
Positive	0.587
Negative	0.593

@@ Line 738: / Line 738: @@
 <p id="pp"> The protocol was improved by using a latin rectangle arrangement in another 96 well plate. A lab robot was used to pipette out the corresponding cultures to their wells according to our preset programme linked to a descrambling spreadsheet. The Latin rectangle is designed so that no sample gets pipetted more than once in any row or column. The order in which the samples get arranged was worked out using the random number function of Microsoft Excel. Sterile LB was loaded into peripheral wells to lessen edge effects which can skew results due to high evaporation at the periphery. These adaptations aim to provide more accurate data by reducing plate effects, samples are spread randomly around the plate so as to minimise differences in results due to irregularities in plate temperature.</p>
-<p id="pp">The measurements were taken using the same calibration settings as before on the Tecan plate reader every hour for 6 hours. The plate was placed into mini shaking incubator at 37°C between plate readings. A scratch was noticed on the plate lid and was replaced. This lid was cold compared to the incubated plate and so condensation formed this could have affected the results. Placed in incubator briefly to warm up. The results were complied into graphs using the iGEM Interlab excel document (Fig. 3 and Fig. 4).</p>
+<div class="col-xs-12" style="margin:0;padding:0;">
+         <img src="https://static.igem.org/mediawiki/2016/2/27/T--Exeter--Interlab-latin_png.png" style="max-width:70%;margin:auto;display:block;">
+<span class="caption">Fig.3</span>
+<br>
+<br>
+</div>
+<p id="pp">The measurements were taken using the same calibration settings as before on the Tecan plate reader every hour for 6 hours. The plate was placed into mini shaking incubator at 37°C between plate readings. A scratch was noticed on the plate lid and was replaced. This lid was cold compared to the incubated plate and so condensation formed this could have affected the results. Placed in incubator briefly to warm up. The results were compiled into graphs using the iGEM Interlab excel document (Fig. 4 and Fig. 5).</p>
 <div class="row">
      <div class="col-xs-6" style="padding:5px 2% 5px 10%;margin:0;">
          <img src="https://static.igem.org/mediawiki/2016/1/1b/T--Exeter--interlabgraphforpart1egl.png"
 		style="max-width:100%;margin:auto;display:block;">
-             <span class="caption">Fig. 3 Fluoresce/Abs 600 measurements using iGEM Interlab layout</span>
+             <span class="caption">Fig. 4 Fluoresce/Abs 600 measurements using iGEM Interlab layout</span>
 		<br>
 		<br>
@@ Line 750: / Line 757: @@
          <img src="https://static.igem.org/mediawiki/2016/a/a6/T--Exeter--latinrectangleinterlabgraphforpart1egl.png"
 		style="max-width:100%;margin:auto;display:block;">
-             <span class="caption">Fig. 4 Fluoresce/Abs 600 measurements using Latin Rectangle layout</span>
+             <span class="caption">Fig. 5 Fluoresce/Abs 600 measurements using Latin Rectangle layout</span>
 		<br>
 		<br>
@@ Line 774: / Line 781: @@
 <p id="pp">An initial OD600 reading was taken using a cuvette reader. Inoculation calculations were then carried out and the cultures of the constructs were grown in new 15ml falcon tubes with 5ml of fresh LB and 5μl of chloramphenicol from a starting OD of 0.02 for 6 hours in a 37°C incubator.</p>
-<p id="pp">The measurements for fluorescence were taken using a BD FACSAria II and the calibration as well as measurement protocols were followed according to the 2016 InterLab Worksheet for Flow Cytometry that is available on the iGEM website. 10,000 events were acquired from calibration beads and from each biological sample. Samples were excited using a 488 nm laser, the filter used was 530/30. Resulting graphs from the FACS measurements are shown below (Fig. 5 - Fig. 10)</p>
+<p id="pp">The measurements for fluorescence were taken using a BD FACSAria II and the calibration as well as measurement protocols were followed according to the 2016 InterLab Worksheet for Flow Cytometry that is available on the iGEM website. 10,000 events were acquired from calibration beads and from each biological sample. Samples were excited using a 488 nm laser, the filter used was 530/30. Resulting graphs from the FACS measurements are shown below (Fig. 6 - Fig. 11)</p>
 <p id="pp">Our results along with the completed worksheet were submitted on the 1/9. No problems were encountered using the protocol or FACS machine. </p>
@@ Line 782: / Line 789: @@
          <img src="https://static.igem.org/mediawiki/2016/7/7b/T--Exeter--facs1.jpg"
 		style="max-width:100%;margin:auto;display:block;">
-             <span class="caption">Fig. 5</span>
+             <span class="caption">Fig. 6</span>
 		<br>
 		<br>
@@ Line 789: / Line 796: @@
          <img src="https://static.igem.org/mediawiki/2016/2/23/T--Exeter--facs2.jpg"
 		style="max-width:100%;margin:auto;display:block;">
-             <span class="caption">Fig. 6</span>
+             <span class="caption">Fig. 7</span>
 		<br>
 		<br>
@@ Line 798: / Line 805: @@
          <img src="https://static.igem.org/mediawiki/2016/5/53/T--Exeter--facsp1.jpg"
 		style="max-width:100%;margin:auto;display:block;">
-             <span class="caption">Fig. 7</span>
+             <span class="caption">Fig. 8</span>
 		<br>
 		<br>
@@ Line 805: / Line 812: @@
          <img src="https://static.igem.org/mediawiki/2016/2/23/T--Exeter--facsp2.jpg"
 		style="max-width:100%;margin:auto;display:block;">
-             <span class="caption">Fig. 8</span>
+             <span class="caption">Fig. 9</span>
 		<br>
 		<br>
@@ Line 814: / Line 821: @@
          <img src="https://static.igem.org/mediawiki/2016/6/6c/T--Exeter--facsp3.jpg"
 		style="max-width:100%;margin:auto;display:block;">
-             <span class="caption">Fig. 9</span>
+             <span class="caption">Fig. 10</span>
 		<br>
 		<br>
@@ Line 821: / Line 828: @@
          <img src="https://static.igem.org/mediawiki/2016/f/f0/T--Exeter--facs0.jpg"
 		style="max-width:100%;margin:auto;display:block;">
-             <span class="caption">Fig. 10</span>
+             <span class="caption">Fig. 11</span>
 		<br>
 		<br>