Team:Sheffield/WLjournal

From 2014.igem.org

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   </head>
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<body> 
<h1> Week 1</h1>
<h1> Week 1</h1>
<h5>Monday 23<sup>rd</sup> June</h5>
<h5>Monday 23<sup>rd</sup> June</h5>
-
Make up 100ml of <a href="#" onClick="MyWindow=window.open('http://imgur.com','MyWindow',
+
Make up 100ml of <a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/prot1','MyWindow',
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');  
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');  
-
return false;" style="color:#DAA520;text-decoration:none">LB Broth</a> and 100ml <a href="#" onClick="MyWindow=window.open('http://imgur.com','MyWindow',
+
return false;" style="color:#DAA520;text-decoration:none">LB Broth</a> and 100ml <a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/prot2','MyWindow',
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');  
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');  
return false;" style="color:#DAA520;text-decoration:none">LB Agar</a>.
return false;" style="color:#DAA520;text-decoration:none">LB Agar</a>.
-
<br><br>Make an <a href="#" onClick="MyWindow=window.open('http://imgur.com','MyWindow',
+
<br><br>Make an <a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/prot3','MyWindow',
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');  
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');  
-
return false;" style="color:#DAA520;text-decoration:none">overnight</a> starter culture of BBa_K258008 to test sterile technique.  
+
return false;" style="color:#DAA520;text-decoration:none">overnight</a> starter culture of <a href="#" onClick="MyWindow=window.
 +
open('https://2014.igem.org/Team:Sheffield/bio1','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">BBa_K258008</a> to test sterile technique.  
<br><h5>Tuesday 24<sup>th</sup> June</h5>
<br><h5>Tuesday 24<sup>th</sup> June</h5>
-
Generate <a href="#" onClick="MyWindow=window.open('http://imgur.com','MyWindow',
+
Generate <a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/prot4','MyWindow',
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');  
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');  
return false;" style="color:#DAA520;text-decoration:none">Chemically competent</a> E.Coli.
return false;" style="color:#DAA520;text-decoration:none">Chemically competent</a> E.Coli.
-
<h4>Make up 8 flasks of 100ml starter culture.
+
<h4><p class="tab">Make up 8 flasks of 100ml starter culture.
<br>4 flasks were checked each time to take the OD readings.
<br>4 flasks were checked each time to take the OD readings.
<br>OD readings to show when the cells are in exponential growth phase and so ready to be made competent.
<br>OD readings to show when the cells are in exponential growth phase and so ready to be made competent.
-
<br><br> TABLE <br>A B C D
+
-
1.25h 0.077 0.078 0.082 0.096
+
-
3.25h 0.567 0.54 0.674 0.543 0.714 0.837 0.809 0.814
+
  <br><br><table style="width:50%; color:#696969">
 +
  <tr>
 +
    <th></th>
 +
    <th>A</th>
 +
<th>B</th>
 +
<th>C</th>
 +
<th>D</th>
 +
  </tr>
 +
  <tr>
 +
    <td>1.25h</td>
 +
    <td>0.077</td>
 +
<td>0.078</td>
 +
<td>0.082</td>
 +
<td>0.096</td>
 +
  </tr>
 +
  <tr>
 +
    <td>3.25h</td>
 +
    <td>0.567</td>
 +
<Td>0.674</td>
 +
<td>0.714</td>
 +
<td>0.809</td>
 +
  </tr>
 +
</table>
 +
 
<br><br>Aliquot into 24 falcon tubes. The cells will need to be spun down in 2 rounds.
<br><br>Aliquot into 24 falcon tubes. The cells will need to be spun down in 2 rounds.
<br>72 eppendorf tubes will be produced, labelled ch. comp, dh5alpha.
<br>72 eppendorf tubes will be produced, labelled ch. comp, dh5alpha.
-
<br>Store at -80&deg;C.</h4>
+
<br>Store at -80&deg;C.</h4></p>
-
</body>
+
 
-
</html>
+
<h1> Week 2 </h1>
 +
<h5>Monday 30<sup>th</sup> June</h5>
 +
 
 +
Make <a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/prot3','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#DAA520;text-decoration:none">overnight</a> starter cultures of the following;
 +
<br><br><br><table style="width:50%; color:#696969">
 +
<tr>
 +
<th>Tube</th>
 +
<th>BioBrick</th>
 +
<th>Antibiotic</th></tr>
 +
<tr><td>1</td> <td><a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bio1','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">BBa_K258008</a></td> <td>Chloramphenicol</td></tr>
 +
<tr><td>2</td> <td><a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bio2','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">BBa_K215107</a></td> <td>Tetracycline</td></tr>
 +
<tr><td>3</td> <td><a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bio3','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">BBa_K861061</a></td> <td>Chloramphenicol</td></tr>
 +
<tr><td>4</td> <td><a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bio4','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">BBa_K215002</a></td> <td>Ampicillin</td></tr>
 +
<tr><td>5</td> <td><a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bio5','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">BBa_K1149021</a></td> <td>Chloramphenicol</td></tr>
 +
<tr><td>6</td> <td><a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bio6','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">BBa_K861060</a></td> <td>Chloramphenicol</td></tr></table>
 +
 
 +
<h4><p class="tab">5&#956;l of an appropriate antibiotic was added to each culture to allow selection for cells containing our biobrick plasmid.</p></h4>
 +
 
 +
 
 +
<h5>Tuesday 1<sup>st</sup> July</h5>
 +
 
 +
Conduct 6 <a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/prot5','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#DAA520;text-decoration:none">mini-preps</a>, one for each of the biobrick cultures, to produce 6 Eppendorfs containing purified DNA.
 +
 
 +
<h5>Wednesday 2<sup>nd</sup> July</h5>
 +
 
 +
<br>Run a <a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/prot6','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#DAA520;text-decoration:none">DNA gel</a> using the purified DNA collected yesterday to check DNA is pure. Use option 3a. 
 +
<Br><h4><p class="tab">Use an 8 well comb to produce the gel.
 +
<br>The table below shows the placement of each DNA sample within the Gel.
 +
<br><br>
 +
<table style="width:50%; color:#696969"><tr><th>Well</th> <th>Biobrick</th> <th>Part Function</th>
 +
<tr><td>1</td>  <td>1kb gene ruler</td><td></td></tr>
 +
<tr><td>2</td> <td><a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bio5','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">BBa_K1149021</a></td> <td>Promoter, RBS, Lipase</td></tr>
 +
<tr><td>3</td> <td><a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bio1','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">BBa_K258008</a></td> <td>ABC transporter</td></tr>
 +
<tr><td>4</td> <td><a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bio6','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">BBa_K861060</a></td> <td>pFadR promoter</td></tr>
 +
<tr><td>5</td> <td><a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bio3','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">BBa_K861061</a></td> <td>pFadR Promoter + RFP</td></tr>
 +
<tr><td>6</td> <td><a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bio2','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">BBa_K215107</a></td> <td>ABC transporter (strong)</td></tr>
 +
<tr><td>7</td> <td><a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bio4','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">BBa_K215002</a></td> <td>pLAC +RBS +secretion signal + streptavidin binding tag</td></tr></table>
 +
 
 +
<br><br>The gel was run at 100v for 45 minutes (due to time constrains).
 +
Below is the image taken of the Gel on the Gel dock.</h4></p>
 +
 
 +
4 <a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/prot7','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#DAA520;text-decoration:none">plates</a> were made, 2 ampicillin and 2 chloramphenicol.
 +
 
 +
<br><br><a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/prot8','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#DAA520;text-decoration:none">Nanodrop</a> the DNA gained from the Mini-prep to generate concentrations of extracted DNA for all 6 biobricks.
 +
 
 +
<br><h4><p class="tab">Results are shown below;
 +
 
 +
</p></h4>
 +
<table style="width:50%; color:#696969"><tr>
 +
<tr> <th>Biobrick</th> <th>Concentration</th></tr>
 +
<tr> <td><a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bio5','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">BBa_K1149021</a></td> <td>115.0</td></tr>
 +
<tr> <td><a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bio1','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">BBa_K258008</a></td> <td>075.9</td></tr>
 +
<tr> <td><a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bio6','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">BBa_K861060</a></td> <td>100.3</td></tr>
 +
<tr> <td><a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bio3','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">BBa_K861061</a></td> <td>130.7</td></tr>
 +
<tr> <td><a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bio2','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">BBa_K215107</a></td> <td>043.3</td></tr>
 +
<tr> <td><a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bio4','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">BBa_K215002</a></td> <td>125.0</td></tr></table>
 +
 
 +
 
 +
<br><a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/prot9','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#DAA520;text-decoration:none">Transform</a> cells to ensuring competency
 +
 
 +
<br><h4><p class="tab">For this transformation We used the BioBricks BBa_K258008 and BBa_K215002 to check if the cells were competent.
 +
<br>Calculations are shown below;
 +
 
 +
<br><br>BBa_K258008
 +
<br>75.9ng x X = 10ng x 100&#956;l
 +
<br>X = 13.2&#956;l
 +
 
 +
<br><br>BBa_K215002
 +
<br>125.0ng x X = 10ng x 100&#956;l
 +
<br>X = 8.0&#956;l</p></h4>
 +
 
 +
 
 +
 
 +
<h5>Friday 4<sup>th</sup> July</h5>
 +
 
 +
The linearised Tlia and KerUS synthesised DNA is moved into a pJet1.2 plasmid using <a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/prot10','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#DAA520;text-decoration:none">blunt end ligation</a>.
 +
 
 +
<br><br>This ligation mix was then used to <a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/prot9','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#DAA520;text-decoration:none">transform</a> 3 eppendorfs of chemically competant cells (made on tuesday 25th);
 +
 
 +
<h4><p class="tab"><li>Into eppendorf 1, pipette 5&#956;l of the ligation mix with the Tlia DNA.
 +
<br><li>Into eppendorf 2, pipette 5&#956;l of the ligation mix containing KerUs DNA.
 +
<br><li>Into eppendorf 3, pipette 5&#956;l of dH<sub>2</sub>O</p></h4>
 +
 
 +
<h4><p class="tab"><br>Incubated for 45 minutes due to time constraints.
 +
<Br>Constructs plated on ampicillin LB to select for the correct colonies.
 +
<br>Plates were checked on saturday AM and ...</p></h4>
 +
 
 +
 
 +
<h1> Week 3 </h1>
 +
<h5>Monday 7<sup>th</sup> July</h5>
 +
 
 +
Make 200ml <a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/prot1','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#DAA520;text-decoration:none">LB Agar</a>and 200ml <a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/prot2','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#DAA520;text-decoration:none">LB Broth</a>.
 +
 
 +
 
 +
<br><br>Make <a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/prot3','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#DAA520;text-decoration:none">overnight</a>
 +
 
 +
cultures of <a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bioT','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">Tlia</a> and <a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bioK','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">KerUS</a>.
 +
<br><br>Make <a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/prot3','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#DAA520;text-decoration:none">overnight</a> cultures of <a href="#" onClick="MyWindow=window.
 +
open('https://2014.igem.org/Team:Sheffield/bioM','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">MC1000</a>.
 +
 
 +
<h5>Tuesday 8<sup>th</sup> July</h5>
 +
 
 +
<a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/prot5','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#DAA520;text-decoration:none">Miniprep</a> the <a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bioT','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">Tlia</a> and <a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/bioK','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#3366ff;text-decoration:none">KerUS</a> cultures to produce purified plasmid DNA.
 +
 
 +
<br><br><a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/prot5','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#DAA520;text-decoration:none">Nanodrop</a> the purified DNA to determine the concentration of DNA.
 +
<h4><p class="tab"> Concentration of DNA from plasmid mini-prep.
 +
<br>TliA - 5.2ng/&#956;l
 +
<br>KerUS - 3.4ng/&#956;l </p></h4>
 +
 
 +
Produce <a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/prot14','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#DAA520;text-decoration:none">glycerol stocks</a> of the Tlia and KerUS cells to be kept as backup.
 +
 
 +
<br><br>Make <a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/prot5','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#DAA520;text-decoration:none">chemically competent</a> MC1000.
 +
<h4><p class="tab">Measure the OD600 of the MC1000 cells using spectrophotometer
 +
<br><br>Flask 1 - 0.803
 +
<br>Flask 2 - 0.624
 +
<br>Blank - 0.066
 +
<br><br>Put 30ml MC1000 into falcon tubes. We have 4 tubes of full MC1000, the 5th one is 21ml MC1000 and 9ml water, the 6th one is all water.</p></h4>
 +
 
 +
<h5>Wednesday 9<sup>th</sup> July</h5>
 +
 
 +
Repeat yesterdays <a href="#" onClick="MyWindow=window.open('https://2014.igem.org/Team:Sheffield/prot5','MyWindow',
 +
'toolbar=no,location=no,directories=no,status=no, menubar=no,scrollbars=no,resizable=no,width=600,height=300');
 +
return false;" style="color:#DAA520;text-decoration:none">miniprep</a> as it didn't yield enough DNA.
 +
<br><h4><p class="tab">Began with two 10ml starter cultures, one of Tlia and one of KerUS.
 +
<br>Decanted by hand into falcon tubes to give four 5ml cultures.</p></h4>
 +
 
 +
</body>
 +
</html>

Latest revision as of 09:08, 13 September 2014

Week 1

Monday 23rd June
Make up 100ml of LB Broth and 100ml LB Agar.

Make an overnight starter culture of BBa_K258008 to test sterile technique.
Tuesday 24th June
Generate Chemically competent E.Coli.

Make up 8 flasks of 100ml starter culture.
4 flasks were checked each time to take the OD readings.
OD readings to show when the cells are in exponential growth phase and so ready to be made competent.

A B C D
1.25h 0.077 0.078 0.082 0.096
3.25h 0.567 0.674 0.714 0.809


Aliquot into 24 falcon tubes. The cells will need to be spun down in 2 rounds.
72 eppendorf tubes will be produced, labelled ch. comp, dh5alpha.
Store at -80°C.

Week 2

Monday 30th June
Make overnight starter cultures of the following;


Tube BioBrick Antibiotic
1 BBa_K258008 Chloramphenicol
2 BBa_K215107 Tetracycline
3 BBa_K861061 Chloramphenicol
4 BBa_K215002 Ampicillin
5 BBa_K1149021 Chloramphenicol
6 BBa_K861060 Chloramphenicol

5μl of an appropriate antibiotic was added to each culture to allow selection for cells containing our biobrick plasmid.

Tuesday 1st July
Conduct 6 mini-preps, one for each of the biobrick cultures, to produce 6 Eppendorfs containing purified DNA.
Wednesday 2nd July

Run a DNA gel using the purified DNA collected yesterday to check DNA is pure. Use option 3a.

Use an 8 well comb to produce the gel.
The table below shows the placement of each DNA sample within the Gel.

Well Biobrick Part Function
1 1kb gene ruler
2 BBa_K1149021 Promoter, RBS, Lipase
3 BBa_K258008 ABC transporter
4 BBa_K861060 pFadR promoter
5 BBa_K861061 pFadR Promoter + RFP
6 BBa_K215107 ABC transporter (strong)
7 BBa_K215002 pLAC +RBS +secretion signal + streptavidin binding tag


The gel was run at 100v for 45 minutes (due to time constrains). Below is the image taken of the Gel on the Gel dock.

4 plates were made, 2 ampicillin and 2 chloramphenicol.

Nanodrop the DNA gained from the Mini-prep to generate concentrations of extracted DNA for all 6 biobricks.

Results are shown below;

Biobrick Concentration
BBa_K1149021 115.0
BBa_K258008 075.9
BBa_K861060 100.3
BBa_K861061 130.7
BBa_K215107 043.3
BBa_K215002 125.0

Transform cells to ensuring competency

For this transformation We used the BioBricks BBa_K258008 and BBa_K215002 to check if the cells were competent.
Calculations are shown below;

BBa_K258008
75.9ng x X = 10ng x 100μl
X = 13.2μl

BBa_K215002
125.0ng x X = 10ng x 100μl
X = 8.0μl

Friday 4th July
The linearised Tlia and KerUS synthesised DNA is moved into a pJet1.2 plasmid using blunt end ligation.

This ligation mix was then used to transform 3 eppendorfs of chemically competant cells (made on tuesday 25th);

  • Into eppendorf 1, pipette 5μl of the ligation mix with the Tlia DNA.
  • Into eppendorf 2, pipette 5μl of the ligation mix containing KerUs DNA.
  • Into eppendorf 3, pipette 5μl of dH2O


    • Incubated for 45 minutes due to time constraints.
    Constructs plated on ampicillin LB to select for the correct colonies.
    Plates were checked on saturday AM and ...

    Week 3

    Monday 7th July
    Make 200ml LB Agarand 200ml LB Broth.

    Make overnight cultures of Tlia and KerUS.

    Make overnight cultures of MC1000.
    Tuesday 8th July
    Miniprep the Tlia and KerUS cultures to produce purified plasmid DNA.

    Nanodrop the purified DNA to determine the concentration of DNA.

    Concentration of DNA from plasmid mini-prep.
    TliA - 5.2ng/μl
    KerUS - 3.4ng/μl

    Produce glycerol stocks of the Tlia and KerUS cells to be kept as backup.

    Make chemically competent MC1000.

    Measure the OD600 of the MC1000 cells using spectrophotometer

    Flask 1 - 0.803
    Flask 2 - 0.624
    Blank - 0.066

    Put 30ml MC1000 into falcon tubes. We have 4 tubes of full MC1000, the 5th one is 21ml MC1000 and 9ml water, the 6th one is all water.

    Wednesday 9th July
    Repeat yesterdays miniprep as it didn't yield enough DNA.

    Began with two 10ml starter cultures, one of Tlia and one of KerUS.
    Decanted by hand into falcon tubes to give four 5ml cultures.

    Retrieved from "http://2014.igem.org/Team:Sheffield/WLjournal"