Team:Exeter/lab book/gibs/wk8

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     <!--Project Division Links-->
     <!--Project Division Links-->
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/proto"; style="color:#57b947">Protocols</a>
 +
      &nbsp;|&nbsp;
       <a href="https://2012.igem.org/Team:Exeter/lab_book/1gp/wk1"; style="color:#57b947">Single Gene Plasmids and Enzyme Characterisation</a>
       <a href="https://2012.igem.org/Team:Exeter/lab_book/1gp/wk1"; style="color:#57b947">Single Gene Plasmids and Enzyme Characterisation</a>
       &nbsp;|&nbsp;
       &nbsp;|&nbsp;
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         -
         -
         </p>
         </p>
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk10"; style="color:#1d1d1b">10th - 14th September</a>
+
         <a href="https://2012.igem.org/Team:Exeter/Results#usepoly"; style="color:#e30614" target="_blank"><font size="3"><b>Results: Part 1</b></font></a>
         <p>
         <p>
         -
         -
         </p>
         </p>
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk11"; style="color:#1d1d1b">17th - 21st September</a>
+
         <a href="https://2012.igem.org/Team:Exeter/Results/GvsB"; style="color:#e30614" target="_blank"><font size="3"><b>Results: Part 2</b></font></a>
       </font>
       </font>
     </div>
     </div>
     <!--End Project Division Week Hyperlinks-->
     <!--End Project Division Week Hyperlinks-->
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     </td>
     </td>
    
    
   <td width="850" height="250">
   <td width="850" height="250">
   <!------------INSERT WEEKLY IMAGE HERE------------>
   <!------------INSERT WEEKLY IMAGE HERE------------>
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     <img src="" alt="" title="" width="850" height="250">
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     <img src="https://static.igem.org/mediawiki/2012/b/b8/Exe2012wbbCPCR.jpg" alt="" title="" width="850" height="250">
   </td>
   </td>
   </tr>
   </tr>
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   <td valign="top" width="850">
   <td valign="top" width="850">
     <div style="text-align:justify">
     <div style="text-align:justify">
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     <font face="DokChampa" color="#1d1d1b" size="2">
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     <font face="Verdana" color="#1d1d1b" size="2">
      
      
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       <font face="DokChampa" color="#57b947" size="4">
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       <font face="Verdana" color="#57b947" size="4">
       <p><b><u>Operon Construction: 27th - 31st August 2012</u></b></p>
       <p><b><u>Operon Construction: 27th - 31st August 2012</u></b></p>
       </font>
       </font>
-
     <!------------ENTER LAB BOOK HERE: PLEASE INCLUDE DATES WITHIN THE WEEK------------>  
+
     <!<p><b>**Tuesday 28.8.12**</b></p><br><p>
 +
 
 +
EcoRI and PstI digest of genes (<i>wbnJ</i>, <i>wbnK</i>, <i>wbbC</i>(d) & <i>wfcA</i>)- for checking on gel</p><p>
 +
 
 +
<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/10" style="color:#57b947"><u>Digestion protocol</u></a></p><p>
 +
Water to 20 µl</p><br><p>
 +
 
 +
3A assembled genes to upstream RBS (BBa_B0034) </p><p>
 +
BBa_B0034 + <i>wbnJ</i> in pSB1K3</p><p>
 +
BBa_B0034 + <i>wbnK</i> in pSB1K3</p><p>
 +
BBa_B0034 + <i>wfcA</i> in pSB1K3</p><p>
 +
BBa_B0034 + <i>wbbC</i>(1) in pSB1K3</p><p>
 +
BBa_B0034 + <i>wbbC</i>(2) in pSB1K3</p><p>
 +
BBa_B0034 + <i>wbbC</i>(3) in pSB1K3</p><br><p>
 +
 
 +
<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/10" style="color:#57b947"><u>3A assembly</u></a></p><p>
 +
Digest concentrations and volumes were altered like so: </p><p>
 +
DNA – 500 ng</p><p>
 +
Enzyme A – 0.5 µl</p><p>
 +
Enzyme B – 0.5 µl</p><p>
 +
NEBuffer 2 – 2 µl</p><p>
 +
BSA – 0.2 µl</p><p>
 +
Water – to 20 µl</p><br><p>
 +
 
 +
Ran gel of PCR product from 24.8.12 (not successful PCR) </p><br><p>
 +
 
 +
PCR using BL21 genomic DNA (extracted previously by Alex B. and Liam) </p><p>
 +
Control - 0.3 µl DNA, 34.7 µl water</p><p>
 +
Genomic – 5 µl DNA, 31 µl water</p><p>
 +
 
 +
<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/12" style="color:#57b947"><u>3 step PCR</u></a></p><p>
 +
<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/7" style="color:#57b947"><u>2 step PCR</u></a></p><p>
 +
PCR setup</p><p>
 +
98°C for 30s</p><p>
 +
98°C for 10s, 50°C for 30s, 72°C for 20s – X 5</p><p>
 +
98°C for 10s, 72°C for 30s – X 25</p><p>
 +
72°C for 10mins</p><p>
 +
Hold temp. 4 °C</p><br><p>
 +
 
 +
Ran PCR gel – success! BL21 genomic DNA was the key. </p><br><p>
 +
 
 +
Transformed extra vectors from plates – standard procedure</p><p>
 +
<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/6" style="color:#57b947"><u>BioBrick extraction</u></a></p><p>
 +
<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/1" style="color:#57b947"><u>Transformation of competent cells</u></a></p><br>
 +
 
 +
 
 +
<b>**Wednesday 29.8.12**</b></p>
 +
 
 +
<p><a href="https://2012.igem.org/Team:Exeter/lab_book/proto/9" style="color:#57b947"><u>PCR purification</u></a> of <i>wbbC</i> PCR product</p>
 +
<p><b><i>Step 1</i></b> 225 µl buffer PB added to 45 µl product</p>
 +
<p><b><i>Additional step after 5</i></b> 750 µl PE added. Centrifuged 1 min, 13000 rpm, rt. Discarded flow through</p>
 +
<p><b><i>Step 7</i></b> 30 µl water added. Stood for 1 minute. 20 µl extra water added (water was not on centre of membrane)</p>
 +
<p>Stored at -20 °C</p><br><p>
 +
 
 +
PCR for Gibson assembly</p><p>
 +
Fragments were primed with overlap primers for making three operon constructs</p><p>
 +
Fragments and volumes (to get 50 ng/µl): </p><p>
 +
Pbad (large) - 1 µl</p><p>
 +
<i>wbnJ</i> – 0.15 µl</p><p>
 +
<i>wbbC</i> - 1 µl</p><p>
 +
<i>wfcA</i> for operons 1 and 3 – 0.15 µl</p><p>
 +
<i>wbnK</i> for 2 and 3 – 0.19 µl</p><p>
 +
Double terminator for 1, 2 and 3 (2 and 3 will be the same, it joins to <i>wbnK</i> in both) – 1 µl</p><p>
 +
(N.B. dilutions of DNA were made to reach appropriate concentration – these dilutions were used for all following PCR reactions which is why each following PCR write-up states 1 µl of DNA was used) </p><p>
 +
water to 50 µl</p><p>
 +
<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/12" style="color:#57b947"><u>3 step PCR</u></a></p><p>
 +
<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/7" style="color:#57b947"><u>2 step PCR</u></a></p><br><p>
 +
PCR setup: </p><p>
 +
98°C for 30s</p><p>
 +
98 °C for 10s, 50°C for 30s, 72°C for 20s – X 5</p><p>
 +
98°C for 10s, 72°C for 30s – X 25</p><p>
 +
72°C for 10mins</p><p>
 +
Hold temp. 4 °C</p><br><p>
 +
 
 +
 
 +
<b>**Thursday 30.8.12**</b></p>
 +
<p>Miniprep of vectors according to <ahref="http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf" style="color:#57B947" target="_blank"><u>Fermentas protocol</u></a></p>
 +
<p><b><i>Step 7</i></b>45 µl water added, followed by another 5 µl</p><br>
 +
 
 +
<p>Gibson PCR</p><p>
 +
Pbad L, <i>wbbC</i>, <i>wfcA</i> 1 & 2, <i>wbnK</i> 1</p><p>
 +
DNA 1 µl, water 33 µl</p><p>
 +
 
 +
<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/12" style="color:#57b947"><u>3 step PCR</u></a></p><p>
 +
PCR setup: </p><p>
 +
98°C for 30s</p><p>
 +
98 °C for 10s, 50°C for 30s, 72°C for 20s – X 5</p><p>
 +
98 °C for 10s, 70°C for 30s, 72°C for 20s – X 5</p><p>
 +
98 °C for 10s, 72°C for 30s – X 20</p><p>
 +
72 °C for 10mins</p><p>
 +
Hold temp. 4 °C</p><br><p>
 +
 
 +
Ran gel – no luck</p>
 +
      
      
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     </font>
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    <a href="https://igem.org/Team.cgi?id=764" style="color:#57B947" target="_blank"><u>Contact Us</u></a>  &nbsp;&nbsp;|&nbsp;&nbsp;
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    <a href="https://2012.igem.org/Team:Exeter/site_map" style="color:#57B947"><u>Site Map</u></a></p>
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Latest revision as of 00:10, 27 September 2012

ExiGEM2012 Lab Book Gibs wk6

Operon Construction: 27th - 31st August 2012

**Tuesday 28.8.12**


EcoRI and PstI digest of genes (wbnJ, wbnK, wbbC(d) & wfcA)- for checking on gel

Digestion protocol

Water to 20 µl


3A assembled genes to upstream RBS (BBa_B0034)

BBa_B0034 + wbnJ in pSB1K3

BBa_B0034 + wbnK in pSB1K3

BBa_B0034 + wfcA in pSB1K3

BBa_B0034 + wbbC(1) in pSB1K3

BBa_B0034 + wbbC(2) in pSB1K3

BBa_B0034 + wbbC(3) in pSB1K3


3A assembly

Digest concentrations and volumes were altered like so:

DNA – 500 ng

Enzyme A – 0.5 µl

Enzyme B – 0.5 µl

NEBuffer 2 – 2 µl

BSA – 0.2 µl

Water – to 20 µl


Ran gel of PCR product from 24.8.12 (not successful PCR)


PCR using BL21 genomic DNA (extracted previously by Alex B. and Liam)

Control - 0.3 µl DNA, 34.7 µl water

Genomic – 5 µl DNA, 31 µl water

3 step PCR

2 step PCR

PCR setup

98°C for 30s

98°C for 10s, 50°C for 30s, 72°C for 20s – X 5

98°C for 10s, 72°C for 30s – X 25

72°C for 10mins

Hold temp. 4 °C


Ran PCR gel – success! BL21 genomic DNA was the key.


Transformed extra vectors from plates – standard procedure

BioBrick extraction

Transformation of competent cells


**Wednesday 29.8.12**

PCR purification of wbbC PCR product

Step 1 225 µl buffer PB added to 45 µl product

Additional step after 5 750 µl PE added. Centrifuged 1 min, 13000 rpm, rt. Discarded flow through

Step 7 30 µl water added. Stood for 1 minute. 20 µl extra water added (water was not on centre of membrane)

Stored at -20 °C


PCR for Gibson assembly

Fragments were primed with overlap primers for making three operon constructs

Fragments and volumes (to get 50 ng/µl):

Pbad (large) - 1 µl

wbnJ – 0.15 µl

wbbC - 1 µl

wfcA for operons 1 and 3 – 0.15 µl

wbnK for 2 and 3 – 0.19 µl

Double terminator for 1, 2 and 3 (2 and 3 will be the same, it joins to wbnK in both) – 1 µl

(N.B. dilutions of DNA were made to reach appropriate concentration – these dilutions were used for all following PCR reactions which is why each following PCR write-up states 1 µl of DNA was used)

water to 50 µl

3 step PCR

2 step PCR


PCR setup:

98°C for 30s

98 °C for 10s, 50°C for 30s, 72°C for 20s – X 5

98°C for 10s, 72°C for 30s – X 25

72°C for 10mins

Hold temp. 4 °C


**Thursday 30.8.12**

Miniprep of vectors according to Fermentas protocol

Step 745 µl water added, followed by another 5 µl


Gibson PCR

Pbad L, wbbC, wfcA 1 & 2, wbnK 1

DNA 1 µl, water 33 µl

3 step PCR

PCR setup:

98°C for 30s

98 °C for 10s, 50°C for 30s, 72°C for 20s – X 5

98 °C for 10s, 70°C for 30s, 72°C for 20s – X 5

98 °C for 10s, 72°C for 30s – X 20

72 °C for 10mins

Hold temp. 4 °C


Ran gel – no luck

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