Team:Exeter/lab book/novpol/wk2

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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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      <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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      <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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      &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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      <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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      &nbsp;|&nbsp;
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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/novpol/wk3"; style="color:#1d1d1b">23rd - 27th July</a>
         <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk3"; style="color:#1d1d1b">23rd - 27th July</a>
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk5"; style="color:#1d1d1b">6th - 10th August</a>
         <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk5"; style="color:#1d1d1b">6th - 10th August</a>
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk6"; style="color:#1d1d1b">13th - 24th August</a>
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk6"; style="color:#1d1d1b">13th - 17th August</a>
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk7"; style="color:#1d1d1b">27th - 31st August</a>
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk7"; style="color:#1d1d1b">20th - 24th August</a>
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        <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk8"; style="color:#1d1d1b">27th - 31st August</a>
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        <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk9"; style="color:#1d1d1b">3rd - 7th September</a>
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        <a href="https://2012.igem.org/Team:Exeter/Results/showcase"; style="color:#e30614" target="_blank"><font size="3"><b>Results</b></font></a>
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   <!------------INSERT WEEKLY IMAGE HERE------------>
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     <!------------ENTER LAB BOOK HERE: PLEASE INCLUDE DATES WITHIN THE WEEK------------>  
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    <p><b>Monday 16th July - Transformation of E coli performed for the ribosome binding site and TetR promoter biobrick.</b></p><br>
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    <p>**<b>Monday 16/07/12</b>**</p><br>
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<p>Performed the <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/6" style="color:#57b947"><u>BioBrick extraction</u></a> of RBS BioBrick (BBa_B0034) and TetR promoter. Subsequently <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/1" style="color:#57b947"><u>Transformed</u></a> the BioBrick onto 50ul of competent cells and plated onto agar containing ampicillin.</p><br>
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<p>**<b>Tuesday 17/07/12</b>**</p><br>
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<p>DNA was re-suspended in 10µL of MilliQ water by pipetting up and down on the well corresponding to the RBS BioBrick (BBa_B0034). The wells containing the biobrick were left for five minutes and then transferred to a labelled Eppendorf tube and spun down briefly in a centrifuge. The suspended RBS DNA (1µL) was pipetted gently into 25µL of <i>E.coli</i> competent cells (Invitrogen), making sure not to mix too rigorously. Competent cells were then incubated on ice for 30 minutes and then heat-shocked for exactly 30 seconds in a 42°C water bath, making sure not to mix or shake. Afterwards, they were quickly placed on ice for 2 minutes. Pre-warmed SOC medium (250µL) was added and then the Eppendorf tube containing the transformed <i>E.coli</i> was secured in a shaking incubator and incubated at 36.8°C for 1 hour at 220rpm.</p><br>
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<p>Cultures containing the RBS and TetR promoter were <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#57b947"><u>transferred</u></a>to liquid medium containing ampicillin.</p><br>
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  <p>**<b>Wednesday 18/07/12</b>**</p><br>
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<p>Whilst incubating, two LB agar plates were made; one would be used for spreading 20µL of transformed <i>E.coli</i> competent cells and the other for 100µL. Therefore 500µL ampicillin was added to 500mL of LB agar to create a 1000-fold dilution.</p><br>
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<p>RBS mini-prep attempt seemed to not pellet so <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#57b947"><u>transferred</u></a> new colonies into liquid broth again containing ampicillin.</p><br>
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<p>**<b>Thursday 19/07/12</b>**</p><br>
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<p>After the transformed <i>E.coli</i> competent cells had finished incubating for an hour, 20 or 100µL of the transformants were spread plated onto LB(amp) agar plates. Remaining transformation mix was stored at 4°C and the two inoculated LB agar plates were inverted and placed in an incubator at 37°C overnight.</p><br>
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<p><b>Tuesday 17th July - Cultures containing the RBS added to liquid medium.</b></p><br>
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<p>Ampicillin, the selection antibiotic, was defrosted. Three isolated colonies from the 100µL spread plate were picked and inoculated in three separate bottles containing 5mL LB broth. 5µL ampicillin was added to each bottle to make a 1000-fold dilution. Each bottle was inverted a couple of times, and then put in a 37°C incubator and left overnight.</p><br>
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<p><b>Wednesday 18th July - RBS mini-prep attempt seemed to not pellet so began transformation procedure again.</b></p><br>
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<p>Overnight cultures were transferred into new Falcon tubes and centrifuged at 3901rcf for 10 minutes at 4°C. It was noticeable that there was no cloudy solution after transferring to new Falcon tubes. Afterwards, when the supernatant was discarded, there was not enough pellet to continue with the mini-prep procedure, and therefore the process of incubating the transformed <i>E.coli</i> overnight in a 37°C incubator was repeated but picking four colonies from the 100µL spread plate instead of three.</p><br>
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<p>Began the transformation procedure (see Monday 16th July) again in case incubation failed to work.</p><br>
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<p><b>Thursday 19th July - Mini-prep and gel electrophoresis of the TetR promoter and RBS.</b></p><br>
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<p>Overnight cultures were transferred into new Falcon tubes and centrifuged at 3900rcf for 2 minutes at 21°C. The supernatant was discarded (being careful not to disturb the pellet). The pellets were re-suspended in 250µL of Re-suspension Buffer by pipetting the solution up and down. Added 250µL of lysis solution and then immediately followed with 350µL of neutralisation buffer. Each Falcon tube was then centrifuged for 5 minutes at 16100rcf at 21°C. Withdrew 850µL of the supernatant and transferred to a geneJET Miniprep column. The column was then centrifuged for 1 minute at 16100rcf at 21°C. The flow-through was discarded (as this contained the sugars, metabolites etc.) and then 500µL of Wash solution was added to the geneJET Miniprep column. This was centrifuged again for 1 minute. Any flow-through was discarded and washed again with extra Wash solution (500µL). This was centrifuged again for 1 minute. The flow-through was emptied and centrifuged again for 1 minute at 21°C with an empty column. The supernatant obtained was transferred to clean, labelled Eppendorfs. 50µL of MilliQ water was added to each Eppendorf and left for a couple of minutes. The concentration of plasmid DNA obtained in each Eppendorf was measured at the Nanodropping machine (in ng/µL, see: Results).</p><br>
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<p>To verify BioBrick parts were cloned successfully, gel electrophoresis was used. Agarose was made (1x 50mL TAE buffer, 0.5g agarose) and then microwaved until the agarose had dissolved. Ethidium bromide (EtBr, 1µL) was added to the agarose gel in the fume cupboard, and then mixed. The EtBr-containing agarose gel was then poured into an electrophoresis plate with a well comb inserted.</p><br>
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<p>The BioBrick parts were removed from the circular plasmids by transferring each cloned plasmid containing different BioBrick parts to different Eppendorf’s containing the Master Mix. This consisted of: 500ng/µL DNA of interest, 1µL EcoR1-HF, 1µL PstI, 5µL 10x NEBuffer2, 0.5µL 100x BSA and enough MilliQ water to make a 50µL total volume. The resulting Master Mix was multiplied by four to compensate for determining the sizes of the four BioBrick parts selected. The DNA-Master Mix solution was left to incubate for 10 minutes at 37°C for the plasmids to be digested.</p><br>
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<p>Loading buffer (4µL) was added to each DNA sample. DNA of each sample (25µL) was added to different wells, including the DNA hyperladder (10µL). Gel electrophoresis was then run for approximately 40 minutes at 150V.</p><br>
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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>Mini-prep</u></a> and gel electrophoresis of the TetR promoter and RBS</p>
 +
<p>To verify BioBrick parts were cloned successfully, gel electrophoresis was used. Agarose was made (1x 50mL TAE buffer, 0.5g agarose and then microwaved until melted). Ethidium bromide (EtBr, 1µL) was added to the agarose gel, and then mixed. The EtBr-containing agarose gel was then poured into an electrophoresis plate with a well comb inserted. The BioBrick parts were removed from the circular plasmids by transferring each cloned plasmid containing different BioBrick parts to different Eppendorfs containing the Master Mix. This consisted of: 500ng/µL RBS DNA, 1µL EcoR1-HF, 1µL PstI, 5µL 10x NEBuffer2, 0.5µL 100x BSA and enough MilliQ H2O to make a 50µL total volume. The resulting Master Mix was multiplied by four to compensate for determining the sizes of the four BioBrick parts. The DNA-Master Mix solution was left to incubate for 10 minutes at 37°C. Loading buffer (4µL) was added to each DNA sample. 25µL of each sample DNA was added to different wells, including DNA hyperladder (10µL). Gel electrophoresis was then run for approximately 40 minutes at 150V.</p><br>
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    <p><u>Website Designed and Built by: Ryan Edginton, James Lynch & Alex Clowsley</u> &nbsp;&nbsp;|&nbsp;&nbsp;
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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 23:57, 26 September 2012

ExiGEM2012 Lab Book NovPol wk2

Showcasing Polysaccharide Production: 16th - 20th July 2012

**Monday 16/07/12**


Performed the BioBrick extraction of RBS BioBrick (BBa_B0034) and TetR promoter. Subsequently Transformed the BioBrick onto 50ul of competent cells and plated onto agar containing ampicillin.


**Tuesday 17/07/12**


Cultures containing the RBS and TetR promoter were transferredto liquid medium containing ampicillin.


**Wednesday 18/07/12**


RBS mini-prep attempt seemed to not pellet so transferred new colonies into liquid broth again containing ampicillin.


**Thursday 19/07/12**


Mini-prep and gel electrophoresis of the TetR promoter and RBS

To verify BioBrick parts were cloned successfully, gel electrophoresis was used. Agarose was made (1x 50mL TAE buffer, 0.5g agarose and then microwaved until melted). Ethidium bromide (EtBr, 1µL) was added to the agarose gel, and then mixed. The EtBr-containing agarose gel was then poured into an electrophoresis plate with a well comb inserted. The BioBrick parts were removed from the circular plasmids by transferring each cloned plasmid containing different BioBrick parts to different Eppendorfs containing the Master Mix. This consisted of: 500ng/µL RBS DNA, 1µL EcoR1-HF, 1µL PstI, 5µL 10x NEBuffer2, 0.5µL 100x BSA and enough MilliQ H2O to make a 50µL total volume. The resulting Master Mix was multiplied by four to compensate for determining the sizes of the four BioBrick parts. The DNA-Master Mix solution was left to incubate for 10 minutes at 37°C. Loading buffer (4µL) was added to each DNA sample. 25µL of each sample DNA was added to different wells, including DNA hyperladder (10µL). Gel electrophoresis was then run for approximately 40 minutes at 150V.

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