Team:Reading/Lab Work

From 2014.igem.org

(Difference between revisions)
Line 49: Line 49:
<td id="15072014">
<td id="15072014">
<br />
<br />
-
<p><font color="#558e2b"><i>15/07/2014</i></font></p>
+
<p><font color="#558e2b"><i>14/07/2014</i></font></p>
<p><font color="#292929">  
<p><font color="#292929">  
-
Did stuff with biobrick. It didn't work. It never works.
+
Set up initial cultures of PCC 7942 according to protocol
 +
<p>Measured OD<sub>750</sub> in order to quantify growth of cyanobacteria. OD measurement preformed according to protocol.</p>
</font></p>
</font></p>
</td>
</td>
Line 60: Line 61:
<p><font color="#558e2b"><i>15/07/2014</i></font></p>
<p><font color="#558e2b"><i>15/07/2014</i></font></p>
<p><font color="#292929">  
<p><font color="#292929">  
-
Did stuff with biobrick. It didn't work. It never works.
+
Following acquisition of DNA ligase we begin BioBrick assembly using protocol available on protocol page.  
 +
<ul>
 +
<li><p>Before assembling the BioBricks we checked the concentration of DNA which were attained from miniprep. Protocol for checking DNA concentration suing Nanodrop is available in protocols section.</p>
 +
<table border="1" style="width:100%">
 +
  <tr>
 +
    <td>Person</td>
 +
    <td>Measurement</td>
 +
    <td>260nm</td>
 +
    <td>280nm</td>
 +
    <td>230nm</td>
 +
    <td>DNAng/ul</td>
 +
  </tr>
 +
  <tr>
 +
    <td>SH</td>
 +
    <td>1</td>
 +
    <td> 0.641 </td>
 +
    <td> 0.356 </td>
 +
    <td> - </td>
 +
    <td> 32.0 </td>
 +
  </tr>
 +
  <tr>
 +
    <td> HC </td>
 +
    <td> 1 </td>
 +
    <td> 0.465 </td>
 +
    <td> 0.261 </td>
 +
    <td> 0.300 </td>
 +
    <td> 23.3 </td>
 +
  </tr>
 +
  <tr>
 +
    <td> </td>
 +
    <td> 2 </td>
 +
    <td> 0.500 </td>
 +
    <td> 0.253 </td>
 +
    <td> 0.294 </td>
 +
    <td> 25.0 </td>
 +
  </tr>
 +
  <tr>
 +
    <td> MS </td>
 +
    <td> 1 </td>
 +
    <td> 0.473 </td>
 +
    <td> 0.240 </td>
 +
    <td> 0.367 </td>
 +
    <td> 23.0 </td>
 +
  </tr>
 +
  <tr>
 +
    <td> </td>
 +
    <td> 2 </td>
 +
    <td> 0.460 </td>
 +
    <td> 0.265 </td>
 +
    <td> 0.340 </td>
 +
    <td> 23.6 </td>
 +
  </tr>
 +
  <tr>
 +
    <td> OS </td>
 +
    <td> 1 </td>
 +
    <td> 0.221 </td>
 +
    <td> 0.120 </td>
 +
    <td> 0.287 </td>
 +
    <td> 11.0 </td>
 +
  </tr>
 +
  <tr>
 +
    <td> </td>
 +
    <td> 2 </td>
 +
    <td> 0.185 </td>
 +
    <td> 0.122 </td>
 +
    <td> 0.307 </td>
 +
    <td> 8.2 </td>
 +
  </tr>
 +
</table>
 +
<li><p>Each sample was measured twice to obtain a more accurate reading.</p>
 +
<ul>
</font></p>
</font></p>
</td>
</td>
<td></td>
<td></td>
-
 
-
<tr>
 
-
<td id="15072014">
 
-
<br />
 
-
<p><font color="#558e2b"><i>14/07/2014</i></font></p>
 
-
<p><font color="#292929">
 
-
Did stuff with biobrick. It didn't work. It never works.
 
-
</font></p>
 
-
</td>
 
-
 
-
<tr>
 
-
<td id="15072014">
 
-
<br />
 
-
<p><font color="#558e2b"><i>15/07/2014</i></font></p>
 
-
<p><font color="#292929">
 
-
Did stuff with biobrick. It didn't work. It never works.
 
-
</font></p>
 
-
</td>
 
<tr>
<tr>
Line 88: Line 141:
<p><font color="#558e2b"><i>17/07/2014</i></font></p>
<p><font color="#558e2b"><i>17/07/2014</i></font></p>
<p><font color="#292929">  
<p><font color="#292929">  
-
Did stuff with biobrick. It didn't work. It never works.
+
<p>Completed BioBrick assembly by transforming competent NEB 5-alpha E. coli. Transformation was preformed according to protocol.</p>
 +
<ul>
 +
<li><p>Bacteria not plated stored at 4℃</p>
 +
</ul>
 +
<p>Prepared selection plates containing ampicillin ready for plating according to protocol</p>
</font></p>
</font></p>
</td>
</td>
Line 97: Line 154:
<p><font color="#558e2b"><i>18/07/2014</i></font></p>
<p><font color="#558e2b"><i>18/07/2014</i></font></p>
<p><font color="#292929">  
<p><font color="#292929">  
-
Did stuff with biobrick. It didn't work. It never works.
+
<p>Checked plates from Biobrick assembly the previous day.</p>
 +
<ul>
 +
  <li><p>No growth seen on an any plates after 18 hours.</p>
 +
  <li><p>Transformed bacteria replated onto regular agar plates and incubated at 37 ℃ to confirm viability of cells.</p>
 +
</ul>
</font></p>
</font></p>
</td>
</td>
Line 106: Line 167:
<p><font color="#558e2b"><i>21/07/2014</i></font></p>
<p><font color="#558e2b"><i>21/07/2014</i></font></p>
<p><font color="#292929">  
<p><font color="#292929">  
-
Did stuff with biobrick. It didn't work. It never works.
+
<p>OD<sub>750</sub> of 7942 cultures from 14/07/2014 measured = 0.080.</p>
 +
<ul>
 +
  <li><p>This media was subcultured using 2 ml of culture into 100 ml of BG11 media into a new 250 ml conical flask.</p>
 +
</ul>
</font></p>
</font></p>
</td>
</td>
Line 114: Line 178:
<br />
<br />
<p><font color="#558e2b"><i>22/07/2014</i></font></p>
<p><font color="#558e2b"><i>22/07/2014</i></font></p>
-
<p><font color="#292929">  
+
<p><font color="#292929">
-
Did stuff with biobrick. It didn't work. It never works.
+
<p>6803 arrived from Howe lab in Cambridge. We also received 3 plasmids for transformation. We have received both wild type (WT) and mutant (terminal oxidase) strains.
 +
<ul>
 +
  <li><p>Started new liquid cultures of 6803 by using 3 loops of bacteria to inoculate 200 ml of BG-11 agar
 +
  <li><p>Created 2 streak plates, one WT and one mutant, on solid BG-11 plates. Protocol for making BG-11 plates is available in protocol section.
 +
  <li><p>Spun down plasmids from Howe lab and stored at -20℃.
 +
</ul>
 +
<p>Checked OD<sub>750</sub> of 7942 cultures = 0.17.
 +
<ul>
 +
  <li><p>Subcultured 7942 with 2 ml from our growing culture and stored it in warm room under light on shaker at 55 rpm.
 +
</ul>
 +
<p>Preformed Gram stain of 6803 using standard protocol and visualised cells under a light microscope.
</font></p>
</font></p>
</td>
</td>
Line 124: Line 198:
<p><font color="#558e2b"><i>23/07/2014</i></font></p>
<p><font color="#558e2b"><i>23/07/2014</i></font></p>
<p><font color="#292929">  
<p><font color="#292929">  
-
Did stuff with biobrick. It didn't work. It never works.
+
<p>Using a new batch of NEB 5-alpha E. coli we retried BioBrick from 17/07/2014.  
 +
<ul>
 +
  <li><p>This time we stored thawed cell on ice rather than in a water bath. Water bath as at ~44℃ rather than the recommended 42℃.
 +
  <li><p>Plated 7 plates of LB agar or Amp LB agar.
 +
</ul>
 +
<p>Checked cultures. Solid 6083 WT showed growth while 7942 subculture did not.
</font></p>
</font></p>
</td>
</td>
Line 133: Line 212:
<p><font color="#558e2b"><i>25/07/2014</i></font></p>
<p><font color="#558e2b"><i>25/07/2014</i></font></p>
<p><font color="#292929">  
<p><font color="#292929">  
-
Did stuff with biobrick. It didn't work. It never works.
+
Checked OD<sub>750</sub> of original 7942 and 6803. Data added to online database.
</font></p>
</font></p>
</td>
</td>
Line 142: Line 221:
<p><font color="#558e2b"><i>28/07/2014</i></font></p>
<p><font color="#558e2b"><i>28/07/2014</i></font></p>
<p><font color="#292929">  
<p><font color="#292929">  
-
Did stuff with biobrick. It didn't work. It never works.
+
<p>Installed new white light.
 +
<p>Checked lux of lamp above the incubator.
</font></p>
</font></p>
</td>
</td>
Line 151: Line 231:
<p><font color="#558e2b"><i>31/07/2014</i></font></p>
<p><font color="#558e2b"><i>31/07/2014</i></font></p>
<p><font color="#292929">  
<p><font color="#292929">  
-
Did stuff with biobrick. It didn't work. It never works.
+
<p>Made a fuel cell using 7942 from a culture from 14/07/2014.  
 +
<ul>
 +
  <li><p>Voltage was measured at 0.2 V however no current was measured
 +
</ul>
</font></p>
</font></p>
</td>
</td>
Line 160: Line 243:
<p><font color="#558e2b"><i>01/08/2014</i></font></p>
<p><font color="#558e2b"><i>01/08/2014</i></font></p>
<p><font color="#292929">  
<p><font color="#292929">  
-
Did stuff with biobrick. It didn't work. It never works.
+
<p>Started new cultures of the WT and mutant 6803 from 22/07/2014 cultures by placing 1 ml of culture into 100 ml of BG-11 media.
 +
<ul>
 +
  <li>Measured OD of these new cultures:
 +
    <ul>
 +
      <li>Original WT = 1.707 Subcultured = 0.019
 +
      <li>Original MT = 1.187 Subcultured = 0.025
 +
    </ul>
 +
</ul>
</font></p>
</font></p>
</td>
</td>
Line 169: Line 259:
<p><font color="#558e2b"><i>04/08/2014</i></font></p>
<p><font color="#558e2b"><i>04/08/2014</i></font></p>
<p><font color="#292929">  
<p><font color="#292929">  
-
Did stuff with biobrick. It didn't work. It never works.
+
<p>Transformed E. coli using 1 plasmid from Cambridge and plated onto amp LB agar plates. Incubated overnight at 37℃.
 +
<p>Send 60% glycerol for autoclaving.
</font></p>
</font></p>
</td>
</td>
Line 178: Line 269:
<p><font color="#558e2b"><i>05/08/2014</i></font></p>
<p><font color="#558e2b"><i>05/08/2014</i></font></p>
<p><font color="#292929">  
<p><font color="#292929">  
-
Did stuff with biobrick. It didn't work. It never works.
+
Checked plates from previous days transformations. Took grown colonies and inoculated LB broth in 4 bijous. These were placed on a shaker at 200 rpm 37℃. Could not add antibiotic (Amp) to the broth as it was not available.
</font></p>
</font></p>
</td>
</td>

Revision as of 23:28, 12 October 2014

University of Reading
Home Team Project Fuel Cell Parts Human Practices Lab book Protocols Attributions


Lab Book

Contents

Here we present our lab book: notes, results and any changes to protocols. These notes are mostly unaltered, presented as they were taken over the course of the lab based aspect of the project. Any alterations were to make them more legible for the web. We hope these brief notes offer insight into the daily goings of our lab team. In addition to this all protocols we used are presented on their own page. The lab book is presented in chronological order. All dates are English format ie. DD/MM/YYYY.

  1. 15th July

14/07/2014

Set up initial cultures of PCC 7942 according to protocol

Measured OD750 in order to quantify growth of cyanobacteria. OD measurement preformed according to protocol.

15/07/2014

Following acquisition of DNA ligase we begin BioBrick assembly using protocol available on protocol page.

  • Before assembling the BioBricks we checked the concentration of DNA which were attained from miniprep. Protocol for checking DNA concentration suing Nanodrop is available in protocols section.

    Person Measurement 260nm 280nm 230nm DNAng/ul
    SH 1 0.641 0.356 - 32.0
    HC 1 0.465 0.261 0.300 23.3
    2 0.500 0.253 0.294 25.0
    MS 1 0.473 0.240 0.367 23.0
    2 0.460 0.265 0.340 23.6
    OS 1 0.221 0.120 0.287 11.0
    2 0.185 0.122 0.307 8.2

  • Each sample was measured twice to obtain a more accurate reading.

17/07/2014

Completed BioBrick assembly by transforming competent NEB 5-alpha E. coli. Transformation was preformed according to protocol.

  • Bacteria not plated stored at 4℃

Prepared selection plates containing ampicillin ready for plating according to protocol

18/07/2014

Checked plates from Biobrick assembly the previous day.

  • No growth seen on an any plates after 18 hours.

  • Transformed bacteria replated onto regular agar plates and incubated at 37 ℃ to confirm viability of cells.

21/07/2014

OD750 of 7942 cultures from 14/07/2014 measured = 0.080.

  • This media was subcultured using 2 ml of culture into 100 ml of BG11 media into a new 250 ml conical flask.

22/07/2014

6803 arrived from Howe lab in Cambridge. We also received 3 plasmids for transformation. We have received both wild type (WT) and mutant (terminal oxidase) strains.

  • Started new liquid cultures of 6803 by using 3 loops of bacteria to inoculate 200 ml of BG-11 agar

  • Created 2 streak plates, one WT and one mutant, on solid BG-11 plates. Protocol for making BG-11 plates is available in protocol section.

  • Spun down plasmids from Howe lab and stored at -20℃.

Checked OD750 of 7942 cultures = 0.17.

  • Subcultured 7942 with 2 ml from our growing culture and stored it in warm room under light on shaker at 55 rpm.

Preformed Gram stain of 6803 using standard protocol and visualised cells under a light microscope.


23/07/2014

Using a new batch of NEB 5-alpha E. coli we retried BioBrick from 17/07/2014.

  • This time we stored thawed cell on ice rather than in a water bath. Water bath as at ~44℃ rather than the recommended 42℃.

  • Plated 7 plates of LB agar or Amp LB agar.

Checked cultures. Solid 6083 WT showed growth while 7942 subculture did not.


25/07/2014

Checked OD750 of original 7942 and 6803. Data added to online database.


28/07/2014

Installed new white light.

Checked lux of lamp above the incubator.


31/07/2014

Made a fuel cell using 7942 from a culture from 14/07/2014.

  • Voltage was measured at 0.2 V however no current was measured


01/08/2014

Started new cultures of the WT and mutant 6803 from 22/07/2014 cultures by placing 1 ml of culture into 100 ml of BG-11 media.

  • Measured OD of these new cultures:
    • Original WT = 1.707 Subcultured = 0.019
    • Original MT = 1.187 Subcultured = 0.025


04/08/2014

Transformed E. coli using 1 plasmid from Cambridge and plated onto amp LB agar plates. Incubated overnight at 37℃.

Send 60% glycerol for autoclaving.


05/08/2014

Checked plates from previous days transformations. Took grown colonies and inoculated LB broth in 4 bijous. These were placed on a shaker at 200 rpm 37℃. Could not add antibiotic (Amp) to the broth as it was not available.


06/08/2014

Did stuff with biobrick. It didn't work. It never works.


07/08/2014

Did stuff with biobrick. It didn't work. It never works.


08/08/2014

Did stuff with biobrick. It didn't work. It never works.


14/08/2014

Did stuff with biobrick. It didn't work. It never works.


15/08/2014

Did stuff with biobrick. It didn't work. It never works.


18/08/2014

Did stuff with biobrick. It didn't work. It never works.


19/08/2014

Did stuff with biobrick. It didn't work. It never works.


20/08/2014

Did stuff with biobrick. It didn't work. It never works.


26/08/2014

Did stuff with biobrick. It didn't work. It never works.


03/09/2014

Did stuff with biobrick. It didn't work. It never works.


05/09/2014

Did stuff with biobrick. It didn't work. It never works.


08/09/2014

Did stuff with biobrick. It didn't work. It never works.


09/09/2014

Did stuff with biobrick. It didn't work. It never works.


10/09/2014

Did stuff with biobrick. It didn't work. It never works.


18/09/2014

Did stuff with biobrick. It didn't work. It never works.


23/09/2014

Did stuff with biobrick. It didn't work. It never works.


24/09/2014

Did stuff with biobrick. It didn't work. It never works.


25/09/2014

Did stuff with biobrick. It didn't work. It never works.


26/09/2014

Did stuff with biobrick. It didn't work. It never works.


29/09/2014

Did stuff with biobrick. It didn't work. It never works.

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Retrieved from "http://2014.igem.org/Team:Reading/Lab_Work"