Team:Exeter/lab book/gibs/wk2

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      <!--Project Division Links-->
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       <a href="https://2012.igem.org/Team:Exeter/lab_book/1gp/wk1"; style="color:#57b947">Single Gene Plasmids and Enzyme Characterisation</a>
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/proto"; style="color:#57b947">Protocols</a>
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        &nbsp;|&nbsp;
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       &nbsp;|&nbsp;
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk1"; style="color:#57b947">Showcasing Polysaccharide Production</a>
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/1gp/wk1"; style="color:#57b947">Single Gene Plasmids and Enzyme Characterisation</a>
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        &nbsp;|&nbsp;
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      &nbsp;|&nbsp;
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/3gip/wk1"; style="color:#57b947">The 3-Gene Inducible Plasmid</a>
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk1"; style="color:#57b947">Showcasing Polysaccharide Production</a>
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        <p>  
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      &nbsp;|&nbsp;
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk1"; style="color:#57b947">Operon Construction</a>       
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/3gip/wk1"; style="color:#57b947">The 3-Gene Inducible Plasmid</a>
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        &nbsp;|&nbsp;
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      <p>  
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/glyco/wk1"; style="color:#57b947">Glycobase</a>
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk1"; style="color:#57b947">Operon Construction</a>       
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        </p>
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      &nbsp;|&nbsp;
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      <!--End Project Division Links-->
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/glyco/wk1"; style="color:#57b947">Glycobase</a>
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      </p>
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk1"; style="color:#1d1d1b">9th - 13th July</a>
         <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk1"; style="color:#1d1d1b">9th - 13th July</a>
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk6"; style="color:#1d1d1b">13th - 24th August</a>
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk6"; style="color:#1d1d1b">13th - 17th August</a>
         <p>
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         -
         -
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         </p>
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk7"; style="color:#1d1d1b">27th - 31st August</a>
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk7"; style="color:#1d1d1b">20th - 24th August</a>
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        <p>
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        -
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        </p>
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        <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk8"; style="color:#1d1d1b">27th - 31st August</a>
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        <p>
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        -
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        </p>
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        <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk9"; style="color:#1d1d1b">3rd - 7th September</a>
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        <p>
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        -
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        </p>
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        <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>
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        <p>
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        -
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        </p>
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        <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>
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   <!------------INSERT WEEKLY IMAGE HERE------------>
   <!------------INSERT WEEKLY IMAGE HERE------------>
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       <p><b><u>Operon Construction: 16th - 20th July 2012</u></b></p>
       <p><b><u>Operon Construction: 16th - 20th July 2012</u></b></p>
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     <!<p><b>16.7.12</b></p>
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     <!<p><b>**Monday 16.7.12**</b></p><br>
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<p>Protocol as on 11.7.12 (basic transformation procedure.)</p>
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<p>TetrRBS and RBS (BBa_B0034) <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/6" style="color:#57b947"><u>BioBricks resuspended</u></a> and used for <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/1" style="color:#1d1d1b"><u>competent cell transformation.</u></a></p>
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<p>TetrRBS and RBS bio brick sequences resuspended in 10µl water</p>
+
<p>1µl of resuspended DNA added to 25µl top10 competent cells.</p>
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<p>Resuspended DNA (1µl) added to top10 competent cells (25µl)</p>
+
<p>LB(amp) (1:1000) plates used for 20µl and 100µl spread plates.</p><br>
-
<p>Incubated on ice for 30 minutes</p>
+
 
-
<p>42 °C heat shock for 30 seconds</p>
+
<p><b>**Tuesday 17.7.12**</b></p><br>
-
<p>Incubated on ice for 2 minutes</p>
+
<p>Previous days spread plate colonies <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#57b947"><u>transferred to liquid medium,</u></a> 1:1000 LB(amp) broth.</p><br>
-
<p>S.O.C. Medium aseptically added (250µl)</p>
+
 
-
<p>Incubated in shaking incubator - 37 C, 220 rpm, 1 hour</p>
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<p><b>**Wednesday 18.7.12**</b></p><br>
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<p>LB(amp) (1:1000) plates made</p>
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<p>Spread plates of 20µl and 100µl transformed E. coli made.</p>
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<p>Overnight incubation at 37 °C</p>
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<p></p>
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<p><b>17.7.12</b></p>
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<p>Made up LB-agar for later use</p>
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<p>Chose 3 colonies from each 100 µl spread plate.</p>
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<p>Colonies transferred by pipette tip into 5 ml LB(amp) broth (1:1000 ratio)</p>
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<p>Incubated at 37 °C, 220 rpm, overnight</p>
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-
<p></p>
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<p><b>18.7.12 (miniprep attempt)</b></p>
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<p>Centrifuged tubes, 4℃, 10 minutes, 3901 rcf</p>
<p>Centrifuged tubes, 4℃, 10 minutes, 3901 rcf</p>
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<p>Pellet was considered too small, probably due to shaking incubator turning off</p>
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<p>Miniprep attempt from <a href="http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf" style="color:#57B947" target="_blank"><u>protocol</u></a></p>
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<p>4 colonies were selected from plates, put in broth and then incubated (as in 17.7.12)</p>
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<p>Pellet was considered too small, believed to be due to shaking incubator turning off overnight</p>
-
<p></p>
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<p>4 colonies were <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#57b947"><u>transferred to liquid medium</u></a> as on previous day</p><br>
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<p><b>19.7.12 (miniprepping)</b></p>
+
 
 +
<p><b>**Thursday 19.7.12**</b></p><br>
<p>Transferred cultures to identical falcon tubes to remove pipette tips</p>
<p>Transferred cultures to identical falcon tubes to remove pipette tips</p>
<p>Centrifuged at 3901 rcf for 5 minutes, 4 °C</p>
<p>Centrifuged at 3901 rcf for 5 minutes, 4 °C</p>
<p>Supernatant tipped away</p>
<p>Supernatant tipped away</p>
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<p>250µl resuspension solution added</p>
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<p> <a href="http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf" style="color:#57B947" target="_blank"><u>Miniprep</u></a> attempt 2</p>
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<p>Pellet resuspended by gentle pipetting up and down</p>
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<p><b><i>Step 10</i></b> 50 µl elution buffer added</p>
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<p>250 µl lysis solution added</p>
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<p>Tubes inverted</p>
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<p>350 µl neutralisation solution rapidly added</p>
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<p>Tubes inverted</p>
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<p>Centrifuged at 13000rpm, 5 minutes, 4 °C</p>
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<p>Supernatant transferred to geneJET miniprep column</p>
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<p>Centrifuged at 13000rpm, 1 minute</p>
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<p>Flow through discarded</p>
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<p>500 µl wash solution added</p>
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<p>Centrifuged at 13000rpm, 30 seconds</p>
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<p>Discard flow through</p>
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<p>500 µl wash solution added</p>
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<p>Centrifuged at 13000rpm, 1 minute</p>
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<p>Discard flow through</p>
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<p>Centrifuged at 13000rpm, 1 minute</p>
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<p>Transferred column to a new 1.5 ml eppendorf</p>
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<p>50 µl elution buffer added</p>
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<p>Incubated for 2 minutes, room temperature</p>
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<p>50 µl elution buffer added</p>
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<p>Incubated for 2 minutes at room temperature</p>
<p>Incubated for 2 minutes at room temperature</p>
<p>Centrifuged for 2 minutes, 13000 rpm</p>
<p>Centrifuged for 2 minutes, 13000 rpm</p>
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<p>Centrifuged for 2 minutes, 13000 rpm</p>
<p>Centrifuged for 2 minutes, 13000 rpm</p>
<p>Columns discarded</p>
<p>Columns discarded</p>
-
<p>Eluted DNA put on ice for analysis with nanodrop spectrophotometer and then stored at -20 C</p>
+
<p>Eluted DNA put on ice for analysis with nanodrop spectrophotometer and then stored at -20 °C</p><br>
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<p>Plasmids digested with following:</p>
+
<p>Plasmids from miniprep digested with EcoR1-HF and PstI according to <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/4" style="color:#57b947"><u>digestion protocol</u></a></p><br>
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<p>Plasmid DNA -  6.5 µl (500ng from largest volume)</p>
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<p><ul>Plasmid DNA -  6.5 µl (500ng from largest volume)</p>
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<p>EcoR1-HF -   1 µl</p>
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<p>EcoR1-HF -       1 µl</p>
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<p>PstI -   1 µl</p>
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<p>PstI -           1 µl</p>
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<p>10x NEBuffer 2 -   5 µl</p>
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<p>10x NEBuffer 2 - 5 µl</p>
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<p>100x BSA -   0.5 µl</p>
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<p>100x BSA -     0.5 µl</p>
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<p>H2O -   36 µl</p>
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<p>MQ Water -           36 µl</ul></p>
-
<p>Incubated for 10 minutes at 37 *C</p>
+
<p>Incubated for 10 minutes at 37 °C</p>
<p>DNA run on gel to test fragments</p>
<p>DNA run on gel to test fragments</p>
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    <p><u>Website Designed and Built by: Ryan Edginton, James Lynch & Alex Clowsley</u> &nbsp;&nbsp;|&nbsp;&nbsp;
 +
    <a href="https://igem.org/Team.cgi?id=764" style="color:#57B947" target="_blank"><u>Contact Us</u></a>  &nbsp;&nbsp;|&nbsp;&nbsp;
 +
    <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 00:09, 27 September 2012

ExiGEM2012 Lab Book Gibs wk2

Operon Construction: 16th - 20th July 2012

 **Monday 16.7.12**


TetrRBS and RBS (BBa_B0034) BioBricks resuspended and used for competent cell transformation.

1µl of resuspended DNA added to 25µl top10 competent cells.

LB(amp) (1:1000) plates used for 20µl and 100µl spread plates.


**Tuesday 17.7.12**


Previous days spread plate colonies transferred to liquid medium, 1:1000 LB(amp) broth.


**Wednesday 18.7.12**


Centrifuged tubes, 4℃, 10 minutes, 3901 rcf

Miniprep attempt from protocol

Pellet was considered too small, believed to be due to shaking incubator turning off overnight

4 colonies were transferred to liquid medium as on previous day


**Thursday 19.7.12**


Transferred cultures to identical falcon tubes to remove pipette tips

Centrifuged at 3901 rcf for 5 minutes, 4 °C

Supernatant tipped away

Miniprep attempt 2

Step 10 50 µl elution buffer added

Incubated for 2 minutes at room temperature

Centrifuged for 2 minutes, 13000 rpm

20 µl milli-Q water added

Centrifuged for 2 minutes, 13000 rpm

Columns discarded

Eluted DNA put on ice for analysis with nanodrop spectrophotometer and then stored at -20 °C


Plasmids from miniprep digested with EcoR1-HF and PstI according to digestion protocol


    Plasmid DNA - 6.5 µl (500ng from largest volume)

    EcoR1-HF - 1 µl

    PstI - 1 µl

    10x NEBuffer 2 - 5 µl

    100x BSA - 0.5 µl

    MQ Water - 36 µl

Incubated for 10 minutes at 37 °C

DNA run on gel to test fragments

Website Designed and Built by: Ryan Edginton, James Lynch & Alex Clowsley   |   Contact Us   |   Site Map

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