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| | <body> | | <body> |
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| − | <!-- Nasal Mucus --> | + | <!-- Nasal Mucus --> |
| | <div> | | <div> |
| | <div class="row"> | | <div class="row"> |
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| − | <p class="journal-content"><i> Removed the signaling peptide from </i> OBP2A in the IDT plasmid </p> | + | <p class="journal-content">Produce a plasmid preperation of <i>OBP2A</i> in the IDT plasmid </p> |
| | </div> | | </div> |
| | <div id="nmcollapsiblew1" class="collapse week-content"> | | <div id="nmcollapsiblew1" class="collapse week-content"> |
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| | <div class="border-day"> | | <div class="border-day"> |
| | <div class="box"> | | <div class="box"> |
| − | <span class="box-title">17/6: Transformation of pIDT-<i>OBP2A</i> in MC1610 <i>e.coli Strain </i></span> | + | <span class="box-title">17/6: Transformation of pIDT-<i>OBP2A</i> in MC1610 <i>e.coli </i>Strain </span> |
| | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#nmcollapsiblew1-1"></span> | | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#nmcollapsiblew1-1"></span> |
| | <div id="nmcollapsiblew1-1" class="collapse box-content"> | | <div id="nmcollapsiblew1-1" class="collapse box-content"> |
| − | <p><b>Aim of Experiment:</b> Transformation of <i>OBP2A</i> from <i>Homo Sapiens</i> in the IDT Plasmid</p> | + | <p><b>Aim of Experiment:</b> To transform pIDT-<i>OBP2A</i> into MC1061 <i> e.coli</i> strain </p> |
| | <p><b>Protocols Used:</b> <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol">Transformation </a> </p> | | <p><b>Protocols Used:</b> <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol">Transformation </a> </p> |
| | <p><b>Results: </b><a data-toggle="modal" data-target="#lbpa-figure1-modal" class="journal-protocol"> N/A </a></p> | | <p><b>Results: </b><a data-toggle="modal" data-target="#lbpa-figure1-modal" class="journal-protocol"> N/A </a></p> |
| − | <p><b>Next Steps:</b> Since the transformation of the <i>OBP2A</i> gene into <i>MC1061</i>e.coli strain requires time to grow the result of this experimentation will not be seen until tomorrow.</p> | + | <p><b>Next Steps:</b> If the transformation is successful the next step will be to produce overnights in preperation for plamsid miniprep. </p> |
| | </div> | | </div> |
| | </div> | | </div> |
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| | <div class="border-day"> | | <div class="border-day"> |
| | <div class="box"> | | <div class="box"> |
| − | <span class="box-title">2/6: Restriction Digests and Ligations</span> | + | <span class="box-title">18/6: Produced overnights from positive colonies of pIDT-<i>OBP2A</i>. </span> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew1-2"></span> | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#nmcollapsiblew1-2"></span> |
| − | <div id="sacollapsiblew1-2" class="collapse box-content"> | + | <div id="nmcollapsiblew1-2" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> To digest <i>LbpA</i> with BamHI and EcoRI and then ligate it into pSB1C3.</p> | + | <p><b>Aim of experiment:</b> To produce overnights of pIDT-<i>OBP2A</i> in MC1061. </p> |
| − | <p><b>Protocols Used:</b> <a data-toggle="modal" data-target="#restrictiondigest-modal" class="journal-protocol">Restriction Digests</a> <a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol"> Ligations</a></p> | + | <p><b>Protocols Used:</b> <a data-toggle="modal" data-target="#onc-modal" class="journal-protocol">Overnight Cultures </a></p> |
| | <p><b>Results:</b> N/A | | <p><b>Results:</b> N/A |
| | | | |
| | | | |
| − | <p><b>Next Steps:</b> Ligations of <i>LbpA</i> + pSB1C3 were left overnight until the next day when they would be transformed into the JM110 strain of <i>E.coli</i>. | + | <p><b>Next Steps:</b> A plasmid preperation of pIDT-<i>OBP2A</i> will be done next. |
| | </div> | | </div> |
| | </div> | | </div> |
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| | <div class="border-day"> | | <div class="border-day"> |
| | <div class="box"> | | <div class="box"> |
| − | <span class="box-title">3/6: Transformations of <i>LpbA</i> + pSB1C3 into JM110 <i>E.coli</i> Strain </span> | + | <span class="box-title">19/6: Plasmid Purification of pIDT-<i>OBP2A</i> overnight culture from yesterday. </span> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew1-3"></span> | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#nmcollapsiblew1-3"></span> |
| − | <div id="sacollapsiblew1-3" class="collapse box-content"> | + | <div id="nmcollapsiblew1-3" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> To transform the pSB1C3 + <i>LbpA</i> ligations into JM110 <i>E.coli</i> cells.</p> | + | <p><b>Aim of experiment:</b> Yesterdays overnight culture was succesful and will now undergo plasmid purification in preperation for use in PCR. </p> |
| − | <p><b>Protocols Used:</b> <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol"> Transformations </a></p> | + | <p><b>Protocols Used:</b> <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Miniprep </a></p> |
| − | <p><b>Results:</b> N/A | + | <p><b>Results:</b> The plasmid preperation yeilded a concentration of 305.53 ng/ul. |
| − | <p><b>Next Steps:</b> Transformations will be checked tomorrow and if successful, overnight cultures will be set up. | + | <p><b>Next Steps:</b> A PCR using the purified pIDT-<i>OBP2A</i> plasmid as the template will be done tomorrow. |
| | | | |
| | </div> | | </div> |
| | </div> | | </div> |
| | </div> | | </div> |
| − | </div> | + | </div> |
| | | | |
| | | | |
| − | | + | </div> |
| − | <div class="row">
| + | |
| − | <div class="border-day">
| + | |
| − | <div class="box">
| + | |
| − | <span class="box-title">4/6: Overnight Cultures</span>
| + | |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew1-4"></span>
| + | |
| − | <div id="sacollapsiblew1-4" class="collapse box-content">
| + | |
| − | <p><b>Aim of experiment:</b> To set up overnight cultures of the <i>LbpA</i> + pSB1C3 JM110 colonies.</p>
| + | |
| − | <p><b>Protocols Used:</b> <a data-toggle="modal" data-target="#onc-modal" class="journal-protocol"> Overnight Cultures
| + | |
| − | </a></p>
| + | |
| − | <p><b>Results:</b> N/A
| + | |
| − | <p><b>Next Steps:</b> A miniprep will be performed on the overnight cultures and a pre-sequence digest test performed tomorrow.
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <div class="row">
| + | |
| − | <div class="border-day">
| + | |
| − | <div class="box">
| + | |
| − | <span class="box-title">5/6: Plasmid Purification and Pre-Sequencing Digest Check</span>
| + | |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew1-5"></span>
| + | |
| − | <div id="sacollapsiblew1-5" class="collapse box-content">
| + | |
| − | <p><b>Aim of experiment:</b> To miniprep the <i>LbpA</i> + pSB1C3 JM110 overnight cultures and perform a pre-sequence restriction digest to check for the presence of the insert.</p>
| + | |
| − | <p><b>Protocols Used:</b> <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Plasmid Purification (QIAprep® Spin Miniprep Kit) </a> <a data-toggle="modal" data-target="#restrictiondigest-modal" class="journal-protocol"> Restriction Digests
| + | |
| − | </a></p>
| + | |
| − | <p><b>Results:</b> N/A
| + | |
| − | <p><b>Next Steps:</b> Since the pre-sequencing digest worked, the miniprep will be sent for sequencing. </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | </div>
| + | |
| − | </div>
| + | |
| | </div> | | </div> |
| | + | </div> |
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| | <div class="border-week"> | | <div class="border-week"> |
| | <div class="box"> | | <div class="box"> |
| − | <div class="labtitle">Week Beginning 8/6/2015</div> | + | <div class="labtitle">Week Beginning 22/6/2015</div> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew2"></span> | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#nmcollapsiblew2"></span> |
| | <div class="box-content"> | | <div class="box-content"> |
| | <p class="journal-summary-heading">Summary</p> | | <p class="journal-summary-heading">Summary</p> |
| | | | |
| | | | |
| − | <p class="journal-content">Sequencing confirmed that <i>LbpA</i> had been successfully cloned into pSB1C3 therefore this week cloning was attempted to put <i>LbpA</i> into the high expression vector pQE80-L.</p> | + | <p class="journal-content"> Removal of the signaling peptide from the start of <i>OBP2A</i> and attempt to insert <i>OBP2A</i> into the biobrick vector pSB1C3. </p> |
| | </div> | | </div> |
| − | <div id="sacollapsiblew2" class="collapse week-content"> | + | <div id="nmcollapsiblew2" class="collapse week-content"> |
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| | </div> | | </div> |
| | </div> | | </div> |
| − | </div> | + | </div> |
| | | | |
| − |
| |
| − | <div class="row">
| |
| − | <div class="border-day">
| |
| − | <div class="box">
| |
| − | <span class="box-title">9/6: Restriction Digests and Ligations of <i>LbpA</i>into pQE80-L</span>
| |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew2-2"></span>
| |
| − | <div id="sacollapsiblew2-2" class="collapse box-content">
| |
| − | <p><b>Aim of experiment:</b>To restrict and ligate the <i>LbpA</i> gene into the pQE80-L plasmid.</p>
| |
| − | <p><b>Protocols Used:</b> <a data-toggle="modal" data-target="#restrictiondigest-modal" class="journal-protocol">Restriction Digests </a> <a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol"> Ligations </a> </p>
| |
| − | <p><b> Results:</b>: N/A </p>
| |
| − | <p><b> Next Steps:</b> Transformations of <i>LbpA</i> + pQE80-L into the JM110 strain will be carried out tomorrow.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
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| | <div class="border-day"> | | <div class="border-day"> |
| | <div class="box"> | | <div class="box"> |
| − | <span class="box-title">10/6: Transformations of <i>LbpA</i> + pQE80-L into JM110</span> | + | <span class="box-title">20/6: PCR of the pIDT-<i>OBP2A</i> plasmid using primers designed for the removal of the signalling peptide. </span> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew2-3"></span> | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#nmcollapsiblew1-4"></span> |
| − | <div id="sacollapsiblew2-3" class="collapse box-content"> | + | <div id="nmcollapsiblew1-4" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> To transform the <i> LbpA</i> + pQE80-L ligations into the E.coli strain JM110.</p> | + | <p><b>Aim of experiment:</b> To set up a PCR using pIDT-<i> OBP2A</i> using priers that if succesful will remove the signalling peptide from the n terminus of <i>OBP2A</i>. </p> |
| − | <p><b>Protocols Used:</b> <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol"> Transformations </a></p> | + | <p><b>Protocols Used:</b> <a data-toggle="modal" data-target="#PCR-modal" class="journal-protocol"> PCR |
| − | <p><b>Results:</b> N/A </p> | + | </a></p> |
| − | <p><b> Next Steps:</b> If the transformations are successful, colonies will be picked to set up overnight cultures. </p> | + | |
| | + | <p><b>Next Steps:</b> The next step will be to gel purify and extract the <i>OBP2A</ i> PCR product from the PCR mixture. Ths will most likely be done tomorrow. |
| | </div> | | </div> |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − | <div class="row">
| |
| − | <div class="border-day">
| |
| − | <div class="box">
| |
| − | <span class="box-title">11/6: Colony PCR of <i>LbpA</i> + pQE80-L Transformation</span>
| |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew2-4"></span>
| |
| − | <div id="sacollapsiblew2-4" class="collapse box-content">
| |
| − | <p><b>Aim of experiment:</b> To set up colony PCR from the single colony obtained from the 2:1 ligation transformation. </p>
| |
| − | <p><b>Protocols Used: </b></p>
| |
| − | <p><b>Results: </b> </p>
| |
| − | <p><b>Next Steps:</b> The gel indicates that the pQE80-L vector has sealed without the presence of the <i>LbpA</i> gene. <i>LbpA</i> will amplified again tomorrow. </p>
| |
| − | </div>
| |
| | </div> | | </div> |
| | </div> | | </div> |
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| | | | |
| − | </div>
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| − | </div>
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| − | </div>
| |
| − | </div>
| |
| | | | |
| − | | + | <div class="row"> |
| − | <!-- WEEK 3 -->
| + | |
| − | | + | |
| − | <div class="row">
| + | |
| − | <div class="border-week">
| + | |
| − | <div class="box">
| + | |
| − | <div class="labtitle">Week Beginning 15/6/15</div>
| + | |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew3"></span>
| + | |
| − | <div class="box-content">
| + | |
| − | <p class="journal-summary-heading">Summary</p>
| + | |
| − | | + | |
| − | | + | |
| − | <p class="journal-content">This week, cloning of <i>LbpA</i> into pQE80-L was continued due to last week's failed attempts.</p>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div id="sacollapsiblew3" class="collapse week-content">
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| | <div class="border-day"> | | <div class="border-day"> |
| | <div class="box"> | | <div class="box"> |
| − | <span class="box-title">15/6: PCR of <i>LbpA</i> for Cloning into pQE80-L</span> | + | <span class="box-title">21/6: Gel extraction of <i>OBP2A</i> from the PCR product and a confirmatory gel image of the purified <i>OBP2A</i>. </span> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew3-1"></span> | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#nmcollapsiblew1-5"></span> |
| − | <div id="sacollapsiblew3-1" class="collapse box-content"> | + | <div id="nmcollapsiblew1-5" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> To re-amplify the <i>LbpA</i> gene for cloning into the pQE80-L vector.</p> | + | <p><b>Aim of experiment:</b> To extract <i>OBP2A</i> from the PCR product using gel extraction. Then to subsequently image the extracted solution to determine the presence of <i>OBP2A<i>. </p> |
| − | <p><b>Protocols Used: </b><a data-toggle="modal" data-target="#PCR-modal" class="journal-protocol"> PCR</a></p> | + | <p><b>Protocols Used:</b> <a data-toggle="modal" data-target="#?-modal" class="journal-protocol"> Gel Extraction |
| − | <p><b>Results:</b> <a data-toggle="modal" data-target="#lbpa-figure3-modal" class="journal-protocol">Figure 3</a></b> </p> | + | </a></p> |
| − | <p><b>Next Steps:</b> The gel shows that <i>LbpA</i> has been successfully amplified so it will be digested and ligated into the pQE80-L vector tomorrow. </p> | + | <p><b>Results:</b> N/A |
| − | </div> | + | <p><b>Next Steps:</b> The gel image shows a band corresponding to the size of <i> OBP2A</i> , The next step will be to restrict this gel extracted <i>OBP2A</ i> in preperation for ligation. </div> |
| | </div> | | </div> |
| | </div> | | </div> |
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| | </div> | | </div> |
| | </div> | | </div> |
| − | </div>
| |
| | </div> | | </div> |
| | | | |
| | | | |
| − | | + | <!-- WEEK 2 --> |
| − | <!-- WEEK 4 --> | + | |
| − | <div class="row"> | + | |
| | <div class="border-week"> | | <div class="border-week"> |
| | <div class="box"> | | <div class="box"> |
| − | <div class="labtitle">Week Beginning 22/6/15</div> | + | <div class="labtitle">Week Beginning 8/6/2015</div> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew4"></span> | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew2"></span> |
| | <div class="box-content"> | | <div class="box-content"> |
| | <p class="journal-summary-heading">Summary</p> | | <p class="journal-summary-heading">Summary</p> |
| | | | |
| | | | |
| − | <p class="journal-content"><i>LbpA</i> was successfully cloned into the high expression vector pQE80-L.</p> | + | <p class="journal-content">Sequencing confirmed that <i>LbpA</i> had been successfully cloned into pSB1C3 therefore this week cloning was attempted to put <i>LbpA</i> into the high expression vector pQE80-L.</p> |
| | </div> | | </div> |
| | + | <div id="sacollapsiblew2" class="collapse week-content"> |
| | | | |
| − | <div id="sacollapsiblew4" class="collapse week-content">
| |
| | | | |
| − | | + | |
| − | | + | |
| | <div class="border-day"> | | <div class="border-day"> |
| | <div class="box"> | | <div class="box"> |
| − | <span class="box-title">23/6: Restriction Digest and Ligation of <i>LbpA</i> into pQE80-L</span> | + | <span class="box-title">8/6: Amplification of <i>LbpA</i> into pQE80-L</span> |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew4-1"></span> | + | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew2-1"></span> |
| − | <div id="sacollapsiblew4-1" class="collapse box-content"> | + | <div id="sacollapsiblew2-1" class="collapse box-content"> |
| − | <p><b>Aim of experiment:</b> To digest the amplified <i>LbpA</i> gene with BamHI and KpnI and subsequently ligate it into the pQE80-L vector.</p> | + | <p><b>Aim of experiment:</b> To amplify <i>LbpA</i> for subsequent cloninng into the pQE80-L vector.</p> |
| − | <p><b>Protocols Used: </b><a data-toggle="modal" data-target="#restrictiondigest-modal" class="journal-protocol"> Restriction Digests </a> <a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol"> Ligations</a></p> | + | <p><b>Protocols Used:</b> <a data-toggle="modal" data-target="#PCR-modal" class="journal-protocol"> PCR </a></p> |
| − | <p><b>Results:</b> N/A </p> | + | <p><b>Results:</b><a data-toggle="modal" data-target="#lbpa-figure2-modal" class="journal-protocol"> Figure 2 </a></p> |
| − | <p><b>Next Steps:</b> The ligations will be transformed into the M15pREP4 strain of <i>E.coli</i>. </p>
| + | <p><b> Next Steps:</b> The PCR product will be digested and ligated into pQE80-L tomorrrow.</p> |
| − | | + | </div> |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="row">
| + | |
| − | <div class="border-day">
| + | |
| − | <div class="box">
| + | |
| − | <span class="box-title">24/6: Transformations of <i>LbpA</i> + pQE80-L into E.coli</span>
| + | |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew4-2"></span>
| + | |
| − | <div id="sacollapsiblew4-2" class="collapse box-content">
| + | |
| − | <p><b>Aim of experiment:</b> To transform the ligations of <i>LbpA</i>+ pQE80-L into the m15pREP4 strain of <i>E.coli.</i></p>
| + | |
| − | <p><b>Protocols Used: </b><a data-toggle="modal" data-target="# transformation- modal" class="journal-protocol"> Transformations</a></p> | + | |
| − | <p><b>Results:</b> N/A </p>
| + | |
| − | <p><b>Next Steps:</b> Overnight cultures will be set up tomorrow if the transformations are successful. </p>
| + | |
| − | | + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="row">
| + | |
| − | <div class="border-day">
| + | |
| − | <div class="box">
| + | |
| − | <span class="box-title">25/6: Overnight Cultures of <i>LbpA</i> + pQE80-L Transformations</span>
| + | |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew4-3"></span>
| + | |
| − | <div id="sacollapsiblew4-3" class="collapse box-content">
| + | |
| − | <p><b>Aim of experiment:</b> To set up overnight cultures of the colonies obtained from the <i>LbpA</i> + pQE80-L transformations.</p>
| + | |
| − | <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#onc-modal" class="journal-protocol">Overnight Cultures </a></p>
| + | |
| − | <p><b>Results:</b> N/A </p>
| + | |
| − | <p><b>Next Steps:</b> Overnight cultures will be miniprepped and a pre-sequence restriction digest will be performed to check for the presence of the <i>LbpA</i> gene. </p> | + | |
| − | | + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div class="row">
| + | |
| − | <div class="border-day">
| + | |
| − | <div class="box">
| + | |
| − | <span class="box-title">26/6: Plasmid Purification of the <i>LbpA</i> + pQE80-L Overnight Cultures</span>
| + | |
| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew4-4"></span>
| + | |
| − | <div id="sacollapsiblew4-4" class="collapse box-content">
| + | |
| − | <p><b>Aim of experiment:</b> To miniprep the overnight cultures from yesterday and perform a pre-sequencing restriction digest using BamHI and KpnI to check for presence of <i>LbpA</i> gene.</p>
| + | |
| − | <p><b>Protocols Used: </b><a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol">Plasmid Purification (QIAprep® Spin Miniprep Kit)</a></p>
| + | |
| − | <p><b>Results:</b> N/A </p>
| + | |
| − | <p><b>Next Steps:</b> A sample of the miniprep was sent for sequencing to confirm the presence of <i>LbpA</i> in the pQE80-L vector. </p>
| + | |
| − | | + | |
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| | </div> | | </div> |
| | </div> | | </div> |
| | </div> | | </div> |
| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| | | | |
| − |
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| − | <!-- WEEK 5 -->
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| − | <div class="row">
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| − | <div class="border-week">
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| − | <div class="box">
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| − | <div class="labtitle">Week Beginning 29/6/15</div>
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| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew5"></span>
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| − | <div class="box-content">
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| − | <p class="journal-summary-heading">Summary</p>
| |
| − |
| |
| − |
| |
| − | <p class="journal-content">Protein expression optimization experiments were performed on LbpA to assay protein production levels. However, cell growth seems to cease when <i>LbpA</i> expression is induced.</p>
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| − | </div>
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| − | <div id="sacollapsiblew5" class="collapse week-content">
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| − | <div class="row">
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| − | <div class="border-day">
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| − | <div class="box">
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| − | <span class="box-title">1/7: <i>LbpA</i> + pQE80-L Overnight Cultures</span>
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| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew5-1"></span>
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| − | <div id="sacollapsiblew5-1" class="collapse box-content">
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| − | <p><b>Aim of experiment:</b> To set up overnight cultures in preparation for protein expression experiments tomorrow.</p>
| |
| − | <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#onc-modal" class="journal-protocol"> Overnight Cultures </a></p>
| |
| − | <p><b>Results:</b> N/A </p>
| |
| − | <p><b>Next Steps:</b> The overnight cultures will be used to set up day cultures tomorrow which will be used to test expression of LbpA. </p>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | <div class="row">
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| − | <div class="border-day">
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| − | <div class="box">
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| − | <span class="box-title">2/7: Optimization of <i>LbpA</i> Expression</span>
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| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew5-2"></span>
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| − | <div id="sacollapsiblew5-2" class="collapse box-content">
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| − | <p><b>Aim of experiment:</b> To set up day cultures and induce expression of <i>LbpA</i> using different concentrations of IPTG and check levels of protein production via SDS-PAGE.</p>
| |
| − | <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#sds-modal" class="journal-protocol"> Protein Expression Optimization </a> </p>
| |
| − | <p><b>Results:</b> N/A</p>
| |
| − | <p><b>Next Steps:</b> The samples were stored in the -20oC freezer overnight and will be ran on a gel tomorrow.</p>
| |
| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | <div class="row">
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| − | <div class="border-day">
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| − | <div class="box">
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| − | <span class="box-title">3/7: Continuation of Optimization of <i>LbpA</i> Expression</span>
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| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew5-3"></span>
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| − | <div id="sacollapsiblew5-3" class="collapse box-content">
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| − | <p><b>Aim of experiment:</b> To test the samples obtained yesterday on an SDS gel.</p>
| |
| − | <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#sds-modal" class="journal-protocol"> Protein Expression Optimization </a> </p>
| |
| − | <p><b>Results:</b> <a data-toggle="modal" data-target="#lbpa-figure4-modal" class="journal-protocol"> Figure 4 </a> </p>
| |
| − | <p><b>Next Steps:</b> Further optimization experiments will be set up next week using different concentrations of IPTG. </p>
| |
| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − |
| |
| − |
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| − | <!-- WEEK 6 -->
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| − | <div class="row">
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| − | <div class="border-week">
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| − | <div class="box">
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| − | <div class="labtitle">Week Beginning 6/7/15</div>
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| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew6"></span>
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| − | <div class="box-content">
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| − | <p class="journal-summary-heading">Summary</p>
| |
| − |
| |
| − |
| |
| − | <p class="journal-content">Further protein expression optimization experiments were set up, this time using a range of IPTG concentrations to induce expression of <i>LbpA</i>.</p>
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| − | </div>
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| − |
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| − | <div id="sacollapsiblew6" class="collapse week-content">
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| − | <div class="row">
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| − | <div class="border-day">
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| − | <div class="box">
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| − | <span class="box-title">7/7: Further Optimization of <i>LbpA</i> Expression</span>
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| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew6-1"></span>
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| − | <div id="sacollapsiblew6-1" class="collapse box-content">
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| − | <p><b>Aim of experiment:</b> To set up further tests to optimize expression of LbpA using different concentrations of IPTG.</p>
| |
| − | <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#sds-modal" class="journal-protocol"> Protein Expression Optimization </a> Note: An SDS gel was not ran, instead the OD<sub>600</sub> readings were recorded for the uninduced and induced cultures at 20 minute intervals for 2 hours, 1 and half hours after the cultures were induced. </p>
| |
| − | <p><b>Results:</b> <a data-toggle="modal" data-target="#lbpa-figure5-modal" class="journal-protocol"> Figure 5 </a> </p>
| |
| − | <p><b>Next Steps:</b> A plate reader experiment will be set up next week to monitor the growth rate of the the cells over a longer period of time after <i>LbpA</i> expression has been induced. </p>
| |
| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − |
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| − | <!-- WEEK 7 -->
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| − | <div class="row">
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| − | <div class="border-week">
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| − | <div class="box">
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| − | <div class="labtitle">Week Beginning 13/7/15</div>
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| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew7"></span>
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| − | <div class="box-content">
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| − | <p class="journal-summary-heading">Summary</p>
| |
| − |
| |
| − |
| |
| − | <p class="journal-content">Protein expression optimization experiments were continued this week. A growth curve assay was carried out to assess the effect of <i>LbpA</i> expression over a longer period of time. </p>
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| − | </div>
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| − |
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| − | <div id="sacollapsiblew7" class="collapse week-content">
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| − | <div class="row">
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| − | <div class="border-day">
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| − | <div class="box">
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| − | <span class="box-title">14/7: Plate Reader Growth Curve Assay</span>
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| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew7-1"></span>
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| − | <div id="sacollapsiblew7-1" class="collapse box-content">
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| − | <p><b>Aim of experiment:</b> To induce expression of <i>LbpA</i> using different concentration of IPTG and allow cells to grow for 16 hours in order to monitor cell growth.</p>
| |
| − | <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#platereader-modal" class="journal-protocol"> Growth Curve Assay </a></p>
| |
| − | <p><b>Results:</b> <a data-toggle="modal" data-target="#lbpa-figure6-modal" class="journal-protocol"> Figure 6 </a> </p>
| |
| − | <p><b>Next Steps:</b> A plate reader experiment will be set up next week to monitor the growth rate of the the cells over a longer period of time after <i>LbpA</i> expression has been induced. </p>
| |
| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − |
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| − | <div class="row">
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| − | <div class="border-day">
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| − | <div class="box">
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| − | <span class="box-title">17/7: Sample Preparation for Western Blot</span>
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| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew7-2"></span>
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| − | <div id="sacollapsiblew7-2" class="collapse box-content">
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| − | <p><b>Aim of experiment:</b> To check if the cells in the induced cultures ~7 hours after being induced are producing LbpA. </p>
| |
| − | <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#sds-modal" class="journal-protocol"> Western Blot Sample Preparation </a> Note: The day cultures were grown for 7 hours after they were induced and then the samples were frozen, to be blotted on Monday. </p>
| |
| − | <p><b>Results:</b> N/A </p>
| |
| − | <p><b>Next Steps:</b> The samples will be ran on an SDS gel on Monday and blotted. </p>
| |
| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − |
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| − | <!-- WEEK 8 -->
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| − | <div class="row">
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| − | <div class="border-week">
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| − | <div class="box">
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| − | <div class="labtitle">Week Beginning 20/7/15</div>
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| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew8"></span>
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| − | <div class="box-content">
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| − | <p class="journal-summary-heading">Summary</p>
| |
| − |
| |
| − |
| |
| − | <p class="journal-content">Samples from the growth curve assay were blotted to see if LbpA is being produced at the later stages of the growth curve. A further growth curve assay was also performed which monitored cell growth until a certain OD<sub>600</sub> was reached, at which point the cultures were induced.</p>
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| − | </div>
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| − |
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| − | <div id="sacollapsiblew8" class="collapse week-content">
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| − | <div class="row">
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| − | <div class="border-day">
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| − | <div class="box">
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| − | <span class="box-title">20/7: Western Blot LpbA Culture Samples</span>
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| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew8-1"></span>
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| − | <div id="sacollapsiblew8-1" class="collapse box-content">
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| − | <p><b>Aim of experiment:</b> To western blot the LbpA culture samples from Friday.</p>
| |
| − | <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#sds-modal" class="journal-protocol"> Western Blot </a></p>
| |
| − | <p><b>Results:</b> Nothing was visible on the blot. </p>
| |
| − | <p><b>Next Steps:</b> Another plate reader experiment will be set up on Thursday where the cultures will be monitored for three hours before induction with IPTG and then for a further 20 hours afterwards. </p>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − |
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| − | <div class="row">
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| − | <div class="border-day">
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| − | <div class="box">
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| − | <span class="box-title">23/7: Growth Curve Assay</span>
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| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew8-2"></span>
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| − | <div id="sacollapsiblew8-2" class="collapse box-content">
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| − | <p><b>Aim of experiment:</b> To check if the cells begin to die after induction with IPTG after having already grown to an OD<sub>600</sub> of 0.5. </p>
| |
| − | <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#platereader2-modal" class="journal-protocol"> Growth Curve Assay </a> </p>
| |
| − | <p><b>Results:</b> <a data-toggle="modal" data-target="#lbpa-figure7-modal" class="journal-protocol"> Figure 7 </a> </p>
| |
| − | <p><b>Next Steps:</b> Samples of these cultures will be taken 6 hours after induction to check for the presence of LbpA. </p>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − |
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| − | <!-- WEEK 9 -->
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| − | <div class="row">
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| − | <div class="border-week">
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| − | <div class="box">
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| − | <div class="labtitle">Week Beginning 27/7/15</div>
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| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew9"></span>
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| − | <div class="box-content">
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| − | <p class="journal-summary-heading">Summary</p>
| |
| − |
| |
| − |
| |
| − | <p class="journal-content">The presence of LbpA was confirmed in the culture 6 hours after induction with IPTG so protein purification was started this week./p>
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| − | </div>
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| − |
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| − | <div id="sacollapsiblew9" class="collapse week-content">
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| − | <div class="row">
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| − | <div class="border-day">
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| − | <div class="box">
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| − | <span class="box-title">27/7: Overnight Cultures</span>
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| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew9-1"></span>
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| − | <div id="sacollapsiblew9-1" class="collapse box-content">
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| − | <p><b>Aim of experiment:</b> To set up a 150ml overnight culture for sub-culturing tomorrow.</p>
| |
| − | <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#onc-modal" class="journal-protocol"> Overnight Cultures </a> Note: 150ml of LB was used and 150µl of each antibiotic. </p>
| |
| − | <p><b>Results:</b> N/A </p>
| |
| − | <p><b>Next Steps:</b>150ml of fresh LB will be inoculated tomorrow with 7.5ml of the overnight cultures. </p>
| |
| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | <div class="row">
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| − | <div class="border-day">
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| − | <div class="box">
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| − | <span class="box-title">28/7: <i>LbpA</i> Expression Assay</span>
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| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew9-2"></span>
| |
| − | <div id="sacollapsiblew9-2" class="collapse box-content">
| |
| − | <p><b>Aim of experiment:</b> To set up day cultures using the overnight cultures from yesterday and test for expression of LbpA 6 hours after induction.</p>
| |
| − | <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#sds-modal" class="journal-protocol"> Protein Expression Test </a> Note: 150ml of fresh LB was innoculated with 7.5ml of the overnight cultures. Only an uninduced control and an 1mM IPTG induced culture was set up. The samples were not blotted, they were only ran on an SDS gel. </p>
| |
| − | <p><b>Results:</b> <a data-toggle="modal" data-target="#lbpa-figure8-modal" class="journal-protocol"> Figure 8 </a> </p>
| |
| − | <p><b>Next Steps:</b> A 3L culture will be set up on Thursday under the same conditions as the overnight cultures in order to try and purify LbpA.</p>
| |
| − | </div>
| |
| − | </div>
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| − | </div>
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| − | </div>
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| − | <div class="row">
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| − | <div class="border-day">
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| − | <div class="box">
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| − | <span class="box-title">29/7: Overnight Cultures for 3L Culture</span>
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| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew9-3"></span>
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| − | <div id="sacollapsiblew9-3" class="collapse box-content">
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| − | <p><b>Aim of experiment:</b> To set up overnight cultures for the 3L day cultures that will be grown tomorrow.</p>
| |
| − | <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#onc-modal" class="journal-protocol"> Overnight Cultures </a> Note: One 150ml overnight culture was set up using 150µl of each antibiotic. </p>
| |
| − | <p><b>Results:</b> N/A </p>
| |
| − | <p><b>Next Steps:</b> The 3L culture will be set up tomorrow allowed to grow to OD<sub>600</sub> of 0.6-1 and then induced with 1mM IPTG and grown for 6 hours.</p>
| |
| − | </div>
| |
| − | </div>
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| − | </div>
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| − | </div>
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| − | <div class="row">
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| − | <div class="border-day">
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| − | <div class="box">
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| − | <span class="box-title">30/7: 3L Culture for Purification of LbpA</span>
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| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew9-4"></span>
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| − | <div id="sacollapsiblew9-4" class="collapse box-content">
| |
| − | <p><b>Aim of experiment:</b> To set up 3L day cultures for purifying LbpA.</p>
| |
| − | <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#pp-modal" class="journal-protocol"> Protein Purification</a> Note: The procedure was stopped at Step 6 and the pellets were frozen at -20<sup>o</sup>C. </p>
| |
| − | <p><b>Results:</b> N/A </p>
| |
| − | <p><b>Next Steps:</b> Protein purification will be continued on Monday.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − |
| |
| − | <!-- WEEK 10 -->
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| − | <div class="row">
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| − | <div class="border-week">
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| − | <div class="box">
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| − | <div class="labtitle">Week Beginning 3/8/15</div>
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| − | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#sacollapsiblew10"></span>
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| − | <div class="box-content">
| |
| − | <p class="journal-summary-heading">Summary</p>
| |
| − |
| |
| − |
| |
| − | <p class="journal-content">The first round of LbpA purification failed at the affinity chromatography stage so another 6L culture was grown this week to attempt purification again.</p>
| |
| − | </div>
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| − |
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| − | <span class="box-title">3/8: Continuation of LbpA Purification</span>
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| − | <p><b>Aim of experiment:</b> To continue with purification of LbpA.</p>
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| − | <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#pp-modal" class="journal-protocol"> Protein Purification</a> Note: The protocol was continued from Step 7</p>
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| − | <p><b>Results:</b> N/A </p>
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| − | <p><b>Next Steps:</b>Size exclusion chromatography was not carried out since an SDS gel showed LbpA was not present after performing affinity chromatography. Another 6L culture will be set up this week.</p>
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| − | <span class="box-title">5/8: Overnight Cultures for 6L Culture</span>
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| − | <p><b>Aim of experiment:</b> To set up overnight cultures for the 6L culture that will be set up tomorrow.</p>
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| − | <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#onc-modal" class="journal-protocol"> Overnight Cultures </a>Note: One 150ml overnight culture was set up using 150µl of each antibiotic.</p>
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| − | <p><b>Results:</b> N/A </p>
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| − | <p><b>Next Steps:</b>The overnight cultures will be used to set up the 6L day culture tomorrow. </p>
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| − | <span class="box-title">6/8: 6L Day Cultures for LbpA Purification</span>
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| − | <p><b>Aim of experiment:</b> To set up 6L day cultures for purifying LbpA and begin the purification process.</p>
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| − | <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#pp-modal" class="journal-protocol"> Protein Purification </a> Note: The protocol was stopped at Step 6.</p>
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| − | <p><b>Results:</b> N/A </p>
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| − | <p><b>Next Steps:</b>Purification of LbpA will be continued on Monday. </p>
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| − | <div class="labtitle">Week Beginning 10/8/15</div>
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| − | <p class="journal-summary-heading">Summary</p>
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| − | <p class="journal-content">heh</p>
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| − | <span class="box-title">10/8: Continuation of LbpA Purification</span>
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| − | <p><b>Aim of experiment:</b> To continue with purification of LbpA.</p>
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| − | <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#pp-modal" class="journal-protocol"> Protein Purification</a> Note: The protocol was continued from Step 7</p>
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| − | <p><b>Results:</b> N/A </p>
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| − | <p><b>Next Steps:</b></p>
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