Team:UCL/Labbook/Week12

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<div class="full_page">
<div class="full_page">
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<p class="body_text"> <a href="https://2013.igem.org/Team:UCL/LabBook/Week1">Week 1</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week2"> Week 2</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week3"> Week 3</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week4"> Week 4</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week5"> Week 5</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week6"> Week 6</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week7"> Week 7</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week8"> Week 8</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week9"> Week 9</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week10"> Week 10</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week11"> Week 11</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week12"> Week 12</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week13"> Week 13</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week14"> Week 14</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week15"> Week 15</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week16"> Week 16</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week17"> Week 17</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week18"> Week 18</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week19"> Week 19</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week20"> Week 20</a>
+
<p class="body_text"> <a href="https://2013.igem.org/Team:UCL/LabBook/Week1">Week 1</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week2"> Week 2</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week3"> Week 3</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week4"> Week 4</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week5"> Week 5</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week6"> Week 6</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week7"> Week 7</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week8"> Week 8</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week9"> Week 9</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week10"> Week 10</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week11"> Week 11</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week12"> Week 12</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week13"> Week 13</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week14"> Week 14</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week15"> Week 15</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week16"> Week 16</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week17"> Week 17</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week18"> Week 18</a>  
</p>  
</p>  
 +
</div>
 +
 +
<p class="minor_title">Week 12</p>
 +
 +
<div class="full_row">
 +
<div class="gap">
 +
</div>
 +
<p class="body_text">
 +
<b>Bacterial Lab</b>
 +
</p>
 +
<p class="body_text">
 +
<b>Monday 19th August</b>
 +
</p>
 +
 +
<p class="body_text">
 +
<a href="http://parts.igem.org/Part:pSB1C3?title=Part:pSB1C3" target="_blank"> pSB1C3</a> <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> streaked</a> plates (well 23.O) were retrieved from 30C incubator. Colony growth visible indicating successful <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> transformation</a>. Colonies were expected to be red in colour, however this was not observed.
 +
</p>
 +
 +
<p class="body_text">
 +
<table>
 +
<tr>
 +
<th>Plate (competent cell vial)</th>
 +
<th>Colony Count</th>
 +
</tr>
 +
<tr>
 +
<td>10</td>
 +
<td>50+</td>
 +
</tr>
 +
<tr>
 +
<td>20</td>
 +
<td>50+</td>
 +
</tr>
 +
</table>
 +
 +
</p>
 +
<p class="body_text">
 +
A single colony from each plate was then inoculated in LB in order to later prepare <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> glycerol stocks</a>.
 +
</p>
 +
<p class="body_text">
 +
iGEM 2012 boxes were searched for <a href="http://parts.igem.org/Part:pSB1C3?title=Part:pSB1C3" target="_blank"> pSB1C3</a>. <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> Transformation</a> was carried out, <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> selective plates</a> were spread, incubated at 37°C overnight.
 +
</p>
 +
<p class="body_text">
 +
Inoculations of the following were carried out in 2mL LB:
 +
</p>
 +
<p class="body_text">
 +
·IRRE+PC+RBS (pSB1C3) (2x in 2mL LB) -> 2ul and 5ul -> incu-shaker
 +
</p>
 +
<p class="body_text">
 +
·RFP+pSB1C3 (scooped from plates) (2x in 2mL LB) -> incu-shaker
 +
</p>
 +
<p class="body_text">
 +
1.5mL miniprep, 0.5mL glycerol stock for following day.
 +
</p>
 +
 +
<p class="body_text">
 +
<b>Tuesday 20th August</b>
 +
</p>
 +
<p class="body_text">
 +
Plates taken from incubation:
 +
</p>
 +
<p class="body_text">
 +
Competent cells transformed with plasmid: Colony growth indicating cells have successfully taken up plasmid.
 +
</p>
 +
<p class="body_text">
 +
Negative control (only competent cells): Colony growth indicating likely problems with <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> chloramphenicol</a>.
 +
</p>
 +
<p class="body_text">
 +
<a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> Chloramphenicol</a> will therefore be re-made.
 +
</p>
 +
 +
<p class="body_text">
 +
<table>
 +
<tr>
 +
<th>Plate (pSB1C3+)</th>
 +
<th>Colony Count</th>
 +
</tr>
 +
<tr>
 +
<td>NUC</td>
 +
<td>5</td>
 +
</tr>
 +
<tr>
 +
<td>LAC</td>
 +
<td>20</td>
 +
</tr>
 +
<tr>
 +
<td>LAC</td>
 +
<td>15</td>
 +
</tr>
 +
<tr>
 +
<td>LAC2</td>
 +
<td>10</td>
 +
</tr>
 +
<tr>
 +
<td>CURL1</td>
 +
<td>30</td>
 +
</tr>
 +
<tr>
 +
<td>Control</td>
 +
<td>50</td>
 +
</tr>
 +
</table>
 +
 +
</p>
 +
<p class="body_text">
 +
take from incu-shaker:
 +
</p>
 +
<p class="body_text">
 +
IRRE+PC+RBS (<a href="http://parts.igem.org/Part:pSB1C3?title=Part:pSB1C3" target="_blank"> pSB1C3</a>) -> 4x 200ul <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> glycerols</a> -> storage at -20°C. 
 +
</p>
 +
<p class="body_text">
 +
RFP+PSB1C3 -> 4x 200ul <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> glycerols</a> -> glycerol storage at -20°C.
 +
</p>
 +
<p class="body_text">
 +
All 4 Falcons were <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> minipreped</a>. Subsequently ran gel showed no bands indicating no DNA presence.
 +
</p>
 +
 +
<p class="body_text">
 +
<table>
 +
<tr>
 +
<th>Item (ul)</th>
 +
<th>IrreAcut</th>
 +
<th>IrreAuc</th>
 +
<th>IrreBcut</th>
 +
<th>IrreBuc</th>
 +
<th>RFPcut</th>
 +
<th>RFPuc</th>
 +
<th>Uncut</th>
 +
<th>Uncut</th>
 +
</tr>
 +
<tr>
 +
<td>pSB1C3</td>
 +
<td>5</td>
 +
<td>5</td>
 +
<td>5</td>
 +
<td>5</td>
 +
<td>5</td>
 +
<td>5</td>
 +
<td>5</td>
 +
<td>5</td>
 +
</tr>
 +
<tr>
 +
<tr>
 +
<td>EcoR1</td>
 +
<td>1</td>
 +
<td>0</td>
 +
<td>1</td>
 +
<td>0</td>
 +
<td>1</td>
 +
<td>0</td>
 +
<td>1</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>Pst1</td>
 +
<td>1</td>
 +
<td>0</td>
 +
<td>1</td>
 +
<td>0</td>
 +
<td>1</td>
 +
<td>0</td>
 +
<td>1</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>Buffer 3</td>
 +
<td>1</td>
 +
<td>0</td>
 +
<td>1</td>
 +
<td>0</td>
 +
<td>1</td>
 +
<td>0</td>
 +
<td>1</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>BSA</td>
 +
<td>0.5</td>
 +
<td>0</td>
 +
<td>0.5</td>
 +
<td>0</td>
 +
<td>0.5</td>
 +
<td>0</td>
 +
<td>0.5</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>dH20</td>
 +
<td>1.5</td>
 +
<td>5</td>
 +
<td>1.5</td>
 +
<td>5</td>
 +
<td>1.5</td>
 +
<td>5</td>
 +
<td>1.5</td>
 +
<td>5</td>
 +
</tr>
 +
<tr>
 +
<th>Total</th>
 +
<td>10</td>
 +
<td>10</td>
 +
<td>10</td>
 +
<td>10</td>
 +
<td>10</td>
 +
<td>10</td>
 +
<td>10</td>
 +
<td>10</td>
 +
</tr>
 +
</table>
 +
 +
</p>
 +
<p class="body_text">
 +
<b>Wednesday 21st August</b>
 +
</p>
 +
<p class="body_text">
 +
Nanodrop of pDNA
 +
</p>
 +
<p class="body_text">
 +
<table>
 +
<tr>
 +
<th>Sample</th>
 +
<th>Concentration (ng/ul)</th>
 +
<th>260/280</th>
 +
</tr>
 +
<tr>
 +
<td>Irre A pDNA</td>
 +
<td>-33.0</td>
 +
<td>1.74</td>
 +
</tr>
 +
<tr>
 +
<td>Irre b pDNA</td>
 +
<td>123.0</td>
 +
<td>0.94</td></tr>
 +
<tr>
 +
<td>GFP pDNA</td>
 +
<td>-52.1</td>
 +
<td>1.63</td>
 +
</tr>
 +
<tr>
 +
<td>LB pDNA</td>
 +
<td>-28.4</td>
 +
<td>1.34</td>
 +
</tr>
 +
</table>
 +
 +
</p>
 +
<p class="body_text">
 +
10x chloramphenicol plates were made using supervisor Yanika Borg's chloramphenicol.
 +
</p>
 +
<p class="body_text">
 +
Meeting with Dr Darren Nesbeth:
 +
</p>
 +
<p class="body_text">
 +
The following Biobricks ordered were brought by our supervisor to be <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> streaked</a> onto the relevant <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> antibiotic plates</a> and inoculated in antibiotic+2mL LB. Incubate at 37°C overnight.
 +
</p>
 +
<p class="body_text">
 +
<table>
 +
<tr>
 +
<th>Biobrick</th>
 +
<th>Plate (+ND)</th>
 +
<th>Inoculation (+ 2mL LB)</th>
 +
</tr>
 +
<tr>
 +
<td>J63008</td>
 +
<td>1X AMP, 1X CMP</td>
 +
<td>2 mL AMP + 2 mL CMP</td>
 +
</tr>
 +
<tr>
 +
<td>K105028</td>
 +
<td>1X AMP</td>
 +
<td>2 mL AMP</td></tr>
 +
<tr>
 +
<td>I712004</td>
 +
<td>1X AMP, 1X CMP</td>
 +
<td>2 mL AMP + 2 mL CMP</td>
 +
</tr>
 +
<tr>
 +
<td>K105030</td>
 +
<td>1X AMP</td>
 +
<td>2 mL AMP</td></tr>
 +
</tr>
 +
<tr>
 +
<td>J63008/9</td>
 +
<td>1X AMP</td>
 +
<td>2 mL AMP</td>
 +
</tr>
 +
<tr>
 +
<td>K105027</td>
 +
<td>1X AMP</td>
 +
<td>2 mL AMP</td></tr>
 +
</tr>
 +
<tr>
 +
<td>K812014</td>
 +
<td>1X AMP, 1X CMP</td>
 +
<td>2 mL AMP + 2 mL CMP</td>
 +
</tr>
 +
</table>
 +
 +
</p>
 +
<p class="body_text">
 +
A <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> PCR</a> of Zeocin was performed, a gel was subsequently ran.
 +
</p>
 +
<p class="body_text">
 +
PCR tube:
 +
</p>
 +
<p class="body_text">
 +
1 - PCR zec bb F, R, 2ul template
 +
</p>
 +
<p class="body_text">
 +
2 - PCR zec bb F, R, 1ul template
 +
</p>
 +
<p class="body_text">
 +
3 - PCR zec F, R, 2ul template
 +
</p>
 +
<p class="body_text">
 +
4 - PCR zec F, R, 1ul template
 +
</p>
 +
<p class="body_text">
 +
5 - PCR negative control zec bb F, R,
 +
</p>
 +
<p class="body_text">
 +
6 - PCR negative control zec F, R
 +
</p>
 +
<p class="body_text">
 +
PCR was unsuccessful.
 +
 +
</p>
 +
<p class="body_text">
 +
<b>Thursday 22nd August</b>
 +
</p>
 +
<p class="body_text">
 +
Results from Biobrick <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> streaking</a> and inoculation:
 +
</p>
 +
 +
<p class="body_text">
 +
<table>
 +
<tr>
 +
<th>Biobrick</th>
 +
<th>Positive Control (ND)</th>
 +
<th>Cmp plate</th>
 +
<th>Amp plate</th>
 +
<th>Falcon</th>
 +
</tr>
 +
<tr>
 +
<td>J63008</td>
 +
<td>Growth</td>
 +
<td>Growth</td>
 +
<td>No Growth</td>
 +
<td>No Growth</td>
 +
</tr>
 +
<tr>
 +
<td>K105028</td>
 +
<td>Growth</td>
 +
<td></td>
 +
<td>Growth</td>
 +
<td>Growth</td>
 +
</tr>
 +
<tr>
 +
<td>K105027</td>
 +
<td>Growth</td>
 +
<td></td>
 +
<td>Growth</td>
 +
<td>Growth</td>
 +
</tr>
 +
<tr>
 +
<td>K105030</td>
 +
<td>Growth</td>
 +
<td></td>
 +
<td>Growth</td>
 +
<td>Growth</td>
 +
</tr>
 +
<tr>
 +
<td>K812014</td>
 +
<td>Growth</td>
 +
<td>Growth</td>
 +
<td></td>
 +
<td>Growth</td>
 +
</tr>
 +
<tr>
 +
<td>J63008/9</td>
 +
<td>Growth</td>
 +
<td></td>
 +
<td>Growth</td>
 +
<td>Growth</td>
 +
</tr>
 +
<tr>
 +
<th>I712004</th>
 +
<td>No Growth</td>
 +
<td>No Growth</td>
 +
<td>No Growth</td>
 +
<td>No Growth</td>
 +
</tr>
 +
</table>
 +
 +
</p>
 +
<p class="body_text">
 +
<a href="https://2013.igem.org/Team:UCL/Project/Protocols"> Glycerol stocks</a> were made from the plates that displayed sufficient colony growth.
 +
</p>
 +
 +
<p class="body_text">
 +
A second attempt at zeocin <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> PCR</a> was performed using Phusion DNA Polymerase.
 +
</p>
 +
<p class="body_text">
 +
1 - Zec bb F, R 2ul
 +
</p>
 +
<p class="body_text">
 +
2 - Zec bb F, R 1ul
 +
</p>
 +
<p class="body_text">
 +
3 - Zec F, R 2ul
 +
</p>
 +
<p class="body_text">
 +
4 - Zec F, R 1ul
 +
</p>
 +
<p class="body_text">
 +
5 - Negative control zec bb F, R
 +
</p>
 +
<p class="body_text">
 +
6 - Negative control zec F, R
 +
</p>
 +
 +
<p class="body_text">
 +
A gel was run with 8ul of each of the 6 reactions. PCR was successful.
 +
</p>
 +
 +
<p class="body_text">
 +
Restriction digest of the following samples:
 +
</p>
 +
<p class="body_text">
 +
A - <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K105028" target="_blank"> K105028</a>
 +
</p>
 +
<p class="body_text">
 +
B - <a href="http://parts.igem.org/Part:BBa_K105027" target="_blank"> K105027</a>
 +
</p>
 +
<p class="body_text">
 +
C - <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K105030" target="_blank"> K105030</a>
 +
</p>
 +
<p class="body_text">
 +
D - <a href="http://parts.igem.org/Part:BBa_K812014" target="_blank"> K812014</a>
 +
</p>
 +
<p class="body_text">
 +
E - <a href="http://parts.igem.org/Part:BBa_J63009" target="_blank"> J63009/8</a>
 +
</p>
 +
 +
<p class="body_text">
 +
<table>
 +
<tr>
 +
<th></th>
 +
<th>Double Digest</th>
 +
<th>Uncut</th>
 +
</tr>
 +
<tr>
 +
<td>Sample</td>
 +
<td>5</td>
 +
<td>5</td>
 +
</tr>
 +
<tr>
 +
<td>EcoR1</td>
 +
<td>1</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>Pst1</td>
 +
<td>1</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>Buffer 3</td>
 +
<td>1</td>
 +
<td>0</td>
 +
</tr>
 +
</tr>
 +
<tr>
 +
<td>BSA</td>
 +
<td>0.5</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>dH2O</td>
 +
<td>1.5</td>
 +
<td>5</td>
 +
</tr>
 +
<tr>
 +
<td>Total</td>
 +
<td>10</td>
 +
<td>10</td>
 +
</tr>
 +
</table>
 +
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/8/89/Aug_21_UCLiGEM2013.jpg');height:488px;width:650px"></div>
 +
<p class="body_text">Nanodrop of samples:</p>
 +
</p>
 +
 +
<p class="body_text">
 +
<table>
 +
<tr>
 +
<th>Sample</th>
 +
<th>260/280</th>
 +
<th>ng/ul</th>
 +
</tr>
 +
<tr>
 +
<td>K105028</td>
 +
<td>2.12</td>
 +
<td>39.0</td>
 +
</tr>
 +
<tr>
 +
<td>K105027</td>
 +
<td>1.99</td>
 +
<td>47.1</td>
 +
</tr>
 +
<tr>
 +
<td>K105030</td>
 +
<td>2.07</td>
 +
<td>48.8</td>
 +
</tr>
 +
<tr>
 +
<td>K812014</td>
 +
<td>1.96</td>
 +
<td>113.3</td>
 +
</tr>
 +
<tr>
 +
<td>J63008/9</td>
 +
<td>1.95</td>
 +
<td>59.7</td>
 +
</tr>
 +
</table>
 +
</p>
 +
 +
<p class="body_text">
 +
PCR Purification
 +
</p>
 +
<p class="body_text">
 +
1 - zeo bb F, R 2ul template
 +
</p>
 +
<p class="body_text">
 +
</p>
 +
<p class="body_text">
 +
2 - zeo bb F, R 1ul template
 +
</p>
 +
<p class="body_text">
 +
^ stored in iGEM 2013 box
 +
</p>
 +
<p class="body_text">
 +
Preparative digest of K812014 (pSB1C3)
 +
</p>
 +
 +
<p class="body_text">
 +
<table>
 +
<tr>
 +
<th>Sample</th>
 +
<th>5ug</th>
 +
</tr>
 +
<tr>
 +
<td>E</td>
 +
<td>7</td>
 +
</tr>
 +
<tr>
 +
<td>P</td>
 +
<td>7</td>
 +
</tr>
 +
<tr>
 +
<td>B3</td>
 +
<td>10</td>
 +
</tr>
 +
<tr>
 +
<td>BSA</td>
 +
<td>2</td>
 +
</tr>
 +
<tr>
 +
<td>dH2O</td>
 +
<td>30</td>
 +
</tr>
 +
<tr>
 +
<td>Total</td>
 +
<td>100</td>
 +
</tr>
 +
</table>
 +
</p>
 +
 +
<p class="body_text">
 +
5ug sample = 44ul of 113.3 ng/ul
 +
</p>
 +
<p class="body_text">
 +
CMV PCR
 +
</p>
 +
<p class="body_text">
 +
Primers used: 2s + 6FW, 2s + bbRE
 +
</p>
 +
 +
<p class="body_text">
 +
<b>Friday 23rd August</b>
 +
</p>
 +
<p class="body_text">
 +
Gel was ran with (lane):
 +
</p>
 +
<p class="body_text">
 +
(3) PCR purified zeo bb FR + 2ul template
 +
</p>
 +
<p class="body_text">
 +
(4) PCR purified zeo bb FR + 1ul template
 +
</p>
 +
<p class="body_text">
 +
(6) bwf + bb RE + 1ul template
 +
</p>
 +
<p class="body_text">
 +
(7) bwf + bb RE + 2ul template
 +
</p>
 +
<p class="body_text">
 +
(8) bwf + bb RE
 +
</p>
 +
<p class="body_text">
 +
Samples in lanes 3 & 4 failed, therefore a nanodrop was recorded:
 +
</p>
 +
 +
<p class="body_text">
 +
<table>
 +
<tr>
 +
<th>Sample</th>
 +
<th>260/280</th>
 +
<th>ng/ul</th>
 +
</tr>
 +
<tr>
 +
<td>PCR purified zeo bb FR + 2ul template</td>
 +
<td>1.66</td>
 +
<td>55.6</td>
 +
</tr>
 +
<tr>
 +
<td>PCR purified zeo bb FR + 1ul template</td>
 +
<td>1.82</td>
 +
<td>17.2</td>
 +
</tr>
 +
</table>
 +
</p>
 +
</div>
 +
 +
<div class="gap">
 +
</div>
 +
 +
<div class="full_row">
 +
<div class="gap">
 +
</div>
 +
<p class="body_text">
 +
<b>Mammalian Lab</b>
 +
 +
</p>
 +
<p class="body_text">
 +
<b>Monday 19th August</b>
 +
Viable cell count data for HeLa growth curve, Day 1 : 0.1 x 10^-6 viable cells per mL
 +
 +
</p>
 +
<p class="body_text">
 +
Disc 1                                                Disc 2
 +
</p>
 +
 +
<p class="body_text">
 +
<table>
 +
</tr>
 +
<th></th>
 +
<th></th>
 +
<th>Disc 1</th>
 +
<th></th>
 +
<th></th>
 +
<th>Disc 2</th>
 +
<th></th>
 +
</tr>
 +
</tr>
 +
<th>Concentration of zeocin (µg/ml)</th>
 +
<th>Confluency (%)</th>
 +
<th>Cell Appearance</th>
 +
<th>Floaters</th>
 +
<th>Confluency (%)</th>
 +
<th>Cell Appearance</th>
 +
<th>Floaters</th>
 +
</tr>
 +
<tr>
 +
<td>0</td>
 +
<td>100</td>
 +
<td>Over confluent</td>
 +
<td>Moderate</td>
 +
<td>100</td>
 +
<td>Over confluent</td>
 +
<td>moderate</td>
 +
</tr>
 +
<tr>
 +
<td>50</td>
 +
<td>50</td>
 +
<td>Moderate swelling and death</td>
 +
<td>Many</td>
 +
<td>80</td>
 +
<td>Moderate swelling and death</td>
 +
<td>Many</td>
 +
</tr>
 +
<tr>
 +
<td>100</td>
 +
<td>20</td>
 +
<td>severe swelling, death</td>
 +
<td>Many</td>
 +
<td>20</td>
 +
<td>severe swelling, death</td>
 +
<td>many</td>
 +
</tr>
 +
<tr>
 +
<td>250</td>
 +
<td>0</td>
 +
<td>-</td>
 +
<td>many</td>
 +
<td>0</td>
 +
<td>-</td>
 +
<td>many</td>
 +
</tr>
 +
<tr>
 +
<td>500</td>
 +
<td>0</td>
 +
<td>-</td>
 +
<td>many</td>
 +
<td>0</td>
 +
<td>-</td>
 +
<td>many</td>
 +
</tr>
 +
<tr>
 +
<td>1000</td>
 +
<td>0</td>
 +
<td>-</td>
 +
<td>many</td>
 +
<td>0</td>
 +
<td>-</td>
 +
<td>many</td>
 +
</tr>
 +
</table>
 +
 +
</p>
 +
<p class="body_text">
 +
Split stock HeLa cells
 +
 +
</p>
 +
<p class="body_text">
 +
<b>Tuesday 20th August</b>
 +
</p>
 +
<p class="body_text">
 +
Viable cell count data for HeLa growth curve, Day 1 : [0.17, 0.068 (anomaly), 0.23] x 10^-6 viable cells per ml
 +
</p>
 +
<p class="body_text">
 +
Disc 1                                                Disc 2
 +
</p>
 +
 +
<p class="body_text">
 +
<table>
 +
</tr>
 +
<th></th>
 +
<th></th>
 +
<th>Disc 1</th>
 +
<th></th>
 +
<th></th>
 +
<th>Disc 2</th>
 +
<th></th>
 +
</tr>
 +
</tr>
 +
<th>Concentration of zeocin (µg/ml)</th>
 +
<th>Confluency (%)</th>
 +
<th>Cell Appearance</th>
 +
<th>Floaters</th>
 +
<th>Confluency (%)</th>
 +
<th>Cell Appearance</th>
 +
<th>Floaters</th>
 +
</tr>
 +
<tr>
 +
<td>0</td>
 +
<td>100</td>
 +
<td>Over confluent and death</td>
 +
<td>Moderate</td>
 +
<td>90</td>
 +
<td>Over confluent</td>
 +
<td>moderate</td>
 +
</tr>
 +
<tr>
 +
<td>50</td>
 +
<td>80</td>
 +
<td>Over confluent, swelling and death</td>
 +
<td>Many</td>
 +
<td>20</td>
 +
<td>Moderate swelling and death</td>
 +
<td>Many</td>
 +
</tr>
 +
<tr>
 +
<td>100</td>
 +
<td>20</td>
 +
<td>severe swelling and death</td>
 +
<td>Many</td>
 +
<td>0</td>
 +
<td>severe swelling, death</td>
 +
<td>many</td>
 +
</tr>
 +
<tr>
 +
<td>250</td>
 +
<td>0</td>
 +
<td>-</td>
 +
<td>many</td>
 +
<td>0</td>
 +
<td>-</td>
 +
<td>many</td>
 +
</tr>
 +
<tr>
 +
<td>500</td>
 +
<td>0</td>
 +
<td>-</td>
 +
<td>many</td>
 +
<td>0</td>
 +
<td>-</td>
 +
<td>many</td>
 +
</tr>
 +
<tr>
 +
<td>1000</td>
 +
<td>0</td>
 +
<td>-</td>
 +
<td>many</td>
 +
<td>0</td>
 +
<td>-</td>
 +
<td>many</td>
 +
</tr>
 +
</table>
 +
</p>
 +
 +
<p class="body_text">
 +
<b>Wednesday 21st August</b>
 +
</p>
 +
<p class="body_text">
 +
Viable cell count data for HeLa growth curve, Day 1 : [0.496, 0.244 (anomaly), 0.356] x 10^-6 viable cells per ml
 +
</p>
 +
 +
<p class="body_text">
 +
<b>Thursday 22nd August</b> - Viable cell count data for HeLa growth curve, Day 1 : [0.79, 1.12, 1.19] x 10^-6 viable cells per ml
 +
</p>
 +
</div>
</div>

Latest revision as of 23:40, 4 October 2013

Lab Weeks

Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16 | Week 17 | Week 18

Week 12

Bacterial Lab

Monday 19th August

pSB1C3 streaked plates (well 23.O) were retrieved from 30C incubator. Colony growth visible indicating successful transformation. Colonies were expected to be red in colour, however this was not observed.

Plate (competent cell vial) Colony Count
10 50+
20 50+

A single colony from each plate was then inoculated in LB in order to later prepare glycerol stocks.

iGEM 2012 boxes were searched for pSB1C3. Transformation was carried out, selective plates were spread, incubated at 37°C overnight.

Inoculations of the following were carried out in 2mL LB:

·IRRE+PC+RBS (pSB1C3) (2x in 2mL LB) -> 2ul and 5ul -> incu-shaker

·RFP+pSB1C3 (scooped from plates) (2x in 2mL LB) -> incu-shaker

1.5mL miniprep, 0.5mL glycerol stock for following day.

Tuesday 20th August

Plates taken from incubation:

Competent cells transformed with plasmid: Colony growth indicating cells have successfully taken up plasmid.

Negative control (only competent cells): Colony growth indicating likely problems with chloramphenicol.

Chloramphenicol will therefore be re-made.

Plate (pSB1C3+) Colony Count
NUC 5
LAC 20
LAC 15
LAC2 10
CURL1 30
Control 50

take from incu-shaker:

IRRE+PC+RBS ( pSB1C3) -> 4x 200ul glycerols -> storage at -20°C.

RFP+PSB1C3 -> 4x 200ul glycerols -> glycerol storage at -20°C.

All 4 Falcons were minipreped. Subsequently ran gel showed no bands indicating no DNA presence.

Item (ul) IrreAcut IrreAuc IrreBcut IrreBuc RFPcut RFPuc Uncut Uncut
pSB1C3 5 5 5 5 5 5 5 5
EcoR1 1 0 1 0 1 0 1 0
Pst1 1 0 1 0 1 0 1 0
Buffer 3 1 0 1 0 1 0 1 0
BSA 0.5 0 0.5 0 0.5 0 0.5 0
dH20 1.5 5 1.5 5 1.5 5 1.5 5
Total 10 10 10 10 10 10 10 10

Wednesday 21st August

Nanodrop of pDNA

Sample Concentration (ng/ul) 260/280
Irre A pDNA -33.0 1.74
Irre b pDNA 123.0 0.94
GFP pDNA -52.1 1.63
LB pDNA -28.4 1.34

10x chloramphenicol plates were made using supervisor Yanika Borg's chloramphenicol.

Meeting with Dr Darren Nesbeth:

The following Biobricks ordered were brought by our supervisor to be streaked onto the relevant antibiotic plates and inoculated in antibiotic+2mL LB. Incubate at 37°C overnight.

Biobrick Plate (+ND) Inoculation (+ 2mL LB)
J63008 1X AMP, 1X CMP 2 mL AMP + 2 mL CMP
K105028 1X AMP 2 mL AMP
I712004 1X AMP, 1X CMP 2 mL AMP + 2 mL CMP
K105030 1X AMP 2 mL AMP
J63008/9 1X AMP 2 mL AMP
K105027 1X AMP 2 mL AMP
K812014 1X AMP, 1X CMP 2 mL AMP + 2 mL CMP

A PCR of Zeocin was performed, a gel was subsequently ran.

PCR tube:

1 - PCR zec bb F, R, 2ul template

2 - PCR zec bb F, R, 1ul template

3 - PCR zec F, R, 2ul template

4 - PCR zec F, R, 1ul template

5 - PCR negative control zec bb F, R,

6 - PCR negative control zec F, R

PCR was unsuccessful.

Thursday 22nd August

Results from Biobrick streaking and inoculation:

Biobrick Positive Control (ND) Cmp plate Amp plate Falcon
J63008 Growth Growth No Growth No Growth
K105028 Growth Growth Growth
K105027 Growth Growth Growth
K105030 Growth Growth Growth
K812014 Growth Growth Growth
J63008/9 Growth Growth Growth
I712004 No Growth No Growth No Growth No Growth

Glycerol stocks were made from the plates that displayed sufficient colony growth.

A second attempt at zeocin PCR was performed using Phusion DNA Polymerase.

1 - Zec bb F, R 2ul

2 - Zec bb F, R 1ul

3 - Zec F, R 2ul

4 - Zec F, R 1ul

5 - Negative control zec bb F, R

6 - Negative control zec F, R

A gel was run with 8ul of each of the 6 reactions. PCR was successful.

Restriction digest of the following samples:

A - K105028

B - K105027

C - K105030

D - K812014

E - J63009/8

Double Digest Uncut
Sample 5 5
EcoR1 1 0
Pst1 1 0
Buffer 3 1 0
BSA 0.5 0
dH2O 1.5 5
Total 10 10

Nanodrop of samples:

Sample 260/280 ng/ul
K105028 2.12 39.0
K105027 1.99 47.1
K105030 2.07 48.8
K812014 1.96 113.3
J63008/9 1.95 59.7

PCR Purification

1 - zeo bb F, R 2ul template

2 - zeo bb F, R 1ul template

^ stored in iGEM 2013 box

Preparative digest of K812014 (pSB1C3)

Sample 5ug
E 7
P 7
B3 10
BSA 2
dH2O 30
Total 100

5ug sample = 44ul of 113.3 ng/ul

CMV PCR

Primers used: 2s + 6FW, 2s + bbRE

Friday 23rd August

Gel was ran with (lane):

(3) PCR purified zeo bb FR + 2ul template

(4) PCR purified zeo bb FR + 1ul template

(6) bwf + bb RE + 1ul template

(7) bwf + bb RE + 2ul template

(8) bwf + bb RE

Samples in lanes 3 & 4 failed, therefore a nanodrop was recorded:

Sample 260/280 ng/ul
PCR purified zeo bb FR + 2ul template 1.66 55.6
PCR purified zeo bb FR + 1ul template 1.82 17.2

Mammalian Lab

Monday 19th August Viable cell count data for HeLa growth curve, Day 1 : 0.1 x 10^-6 viable cells per mL

Disc 1 Disc 2

Disc 1 Disc 2
Concentration of zeocin (µg/ml) Confluency (%) Cell Appearance Floaters Confluency (%) Cell Appearance Floaters
0 100 Over confluent Moderate 100 Over confluent moderate
50 50 Moderate swelling and death Many 80 Moderate swelling and death Many
100 20 severe swelling, death Many 20 severe swelling, death many
250 0 - many 0 - many
500 0 - many 0 - many
1000 0 - many 0 - many

Split stock HeLa cells

Tuesday 20th August

Viable cell count data for HeLa growth curve, Day 1 : [0.17, 0.068 (anomaly), 0.23] x 10^-6 viable cells per ml

Disc 1 Disc 2

Disc 1 Disc 2
Concentration of zeocin (µg/ml) Confluency (%) Cell Appearance Floaters Confluency (%) Cell Appearance Floaters
0 100 Over confluent and death Moderate 90 Over confluent moderate
50 80 Over confluent, swelling and death Many 20 Moderate swelling and death Many
100 20 severe swelling and death Many 0 severe swelling, death many
250 0 - many 0 - many
500 0 - many 0 - many
1000 0 - many 0 - many

Wednesday 21st August

Viable cell count data for HeLa growth curve, Day 1 : [0.496, 0.244 (anomaly), 0.356] x 10^-6 viable cells per ml

Thursday 22nd August - Viable cell count data for HeLa growth curve, Day 1 : [0.79, 1.12, 1.19] x 10^-6 viable cells per ml

Retrieved from "http://2013.igem.org/Team:UCL/Labbook/Week12"