Difference between revisions of "Team:Dundee/lucylabjournal"
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<div id="collapsiblew5-3" class="collapse box-content"> | <div id="collapsiblew5-3" class="collapse box-content"> | ||
<p><b>Aim of experiment:</b> To test the samples obtained yesterday on an SDS gel.</p> | <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>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>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> | <p><b>Next Steps:</b> Further optimization experiments will be set up next week using different concentrations of IPTG. </p> | ||
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<div id="collapsiblew6-1" class="collapse box-content"> | <div id="collapsiblew6-1" class="collapse box-content"> | ||
<p><b>Aim of experiment:</b> To set up further tests to optimize expression of LbpA using different concentrations of IPTG.</p> | <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 OD600 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>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 OD600 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>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 LbpA expression has been induced. </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 LbpA expression has been induced. </p> | ||
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<div id="collapsiblew7-2" class="collapse box-content"> | <div id="collapsiblew7-2" class="collapse box-content"> | ||
<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>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>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>Results:</b> N/A </p> | ||
<p><b>Next Steps:</b> The samples will be ran on an SDS gel on Monday and blotted. </p> | <p><b>Next Steps:</b> The samples will be ran on an SDS gel on Monday and blotted. </p> | ||
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<div id="collapsiblew8-1" class="collapse box-content"> | <div id="collapsiblew8-1" class="collapse box-content"> | ||
<p><b>Aim of experiment:</b> To western blot the LbpA culture samples from Friday.</p> | <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>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>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> | <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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</p> | </p> | ||
<p> | <p> | ||
− | Once in the developing room, the film was exposed to the blot for 30 seconds, 2 minutes and 5 minutes and then placed in the film developer. | + | 16. Once in the developing room, the film was exposed to the blot for 30 seconds, 2 minutes and 5 minutes and then placed in the film developer. |
</p> | </p> | ||
Revision as of 21:10, 8 August 2015
LABJOURNAL
BioSpray
Our forensic toolkit aims to use synthetic biology approaches to improve on the current methods used by crime scene investigators.
Summary
Lactoferrin binding protein A (LbpA) was successfully cloned into the vector pSB1C3.
Aim of experiment: To digest LbpA with BamHI and EcoRI and then ligate it into pSB1C3.
Protocols Used: Restriction Digests Ligations
Results: N/A
Next Steps: Ligations of LbpA + pSB1C3 were left overnight until the next day when they would be transformed into JM110 cells.
Aim of experiment: To transform the pSB1C3 + LbpA ligations into JM110 cells.
Protocols Used: Transformations
Results: N/A
Next Steps: Transformations will be checked tomorrow and if successful, overnight cultures will be set up.
Aim of experiment: To set up overnight cultures of the LbpA + pSB1C3 JM110 colonies.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: A miniprep will be performed on the overnight cultures and a pre-sequence digest test performed tomorrow.
Aim of experiment: To miniprep the LbpA + pSB1C3 JM110 overnight cultures and perform a pre-sequence restriction digest to check for the presence of the insert.
Protocols Used: Plasmid Purification (QIAprep® Spin Miniprep Kit) Restriction Digests
Results: N/A
Next Steps: Since the pre-sequencing digest worked, the miniprep will be sent for sequencing.
Week summary
faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. .
Aim of experiment:To restrict and ligate the LbpA gene into the pQE80-L plasmid.
Protocols Used: Restriction Digests Ligations
Results:: N/A
Next Steps: Transformations of LbpA + pQE80-L into the JM110 strain will be carried out tomorrow.
Aim of experiment: To transform the LbpA + pQE80-L ligations into the E.coli strain JM110.
Protocols Used: Transformations
Results: N/A
Next Steps: If the transformations are successful, colonies will be picked to set up overnight cultures.
Aim of experiment: To set up colony PCR from the single colony obtained from the 2:1 ligation transformation.
Protocols Used:
Results:
Next Steps: The gel indicates that the pQE80-L vector has sealed without the presence of the LbpA gene. LbpA will amplified again tomorrow.
Week summary
faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. .
Week summary
faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. .
Aim of experiment: To digest the amplified LbpA gene with BamHI and KpnI and subsequently ligate it into the pQE80-L vector.
Protocols Used: Restriction Digests Ligations
Results: N/A
Next Steps: The ligations will be transformed into the M15pREP4 strain of E.coli.
Aim of experiment: To transform the ligations of LbpA + pQE80-L into the m15pREP4 strain of E.coli.
Protocols Used: Transformations
Results: N/A
Next Steps: Overnight cultures will be set up tomorrow if the transformations are successful.
Aim of experiment: To set up overnight cultures of the colonies obtained from the LbpA + pQE80-L transformations.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: Overnight cultures will be miniprepped and a pre-sequence restriction digest will be performed to check for the presence of the LbpA gene.
Aim of experiment: To miniprep the overnight cultures from yesterday and perform a pre-sequencing restriction digest using BamHI and KpnI to check for presence of LbpA gene.
Protocols Used: Plasmid Purification (QIAprep® Spin Miniprep Kit)
Results: N/A
Next Steps: A sample of the miniprep was sent for sequencing to confirm the presence of LbpA in the pQE80-L vector.
Week summary
faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. .
Aim of experiment: To set up overnight cultures in preparation for protein expression experiments tomorrow.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: The overnight cultures will be used to set up day cultures tomorrow which will be used to test expression of the LbpA.
Aim of experiment: To set up day cultures and induce expression of LbpA using different concentrations of IPTG and check levels of protein expression via SDS-PAGE.
Protocols Used: Protein Expression Optimization
Results: N/A
Next Steps: The samples were stored in the -20oC freezer overnight and will be ran on a gel tomorrow.
Aim of experiment: To test the samples obtained yesterday on an SDS gel.
Protocols Used: Protein Expression Optimization
Results: Figure 4
Next Steps: Further optimization experiments will be set up next week using different concentrations of IPTG.
Week summary
faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. .
Aim of experiment: To set up further tests to optimize expression of LbpA using different concentrations of IPTG.
Protocols Used: Protein Expression Optimization Note: An SDS gel was not ran, instead the OD600 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.
Results: Figure 5
Next Steps: 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 LbpA expression has been induced.
Week summary
faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. .
Aim of experiment: To induce expression of LbpA using different concentration of IPTG and allow cells to grow for 16 hours in order to monitor cell growth.
Protocols Used: Growth Curve Assay
Results: Figure 6
Next Steps: 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 LbpA expression has been induced.
Aim of experiment: To check if the cells in the induced cultures ~7 hours after being induced are producing LbpA.
Protocols Used: Western Blot Sample Preparation Note: The day cultures were grown for 7 hours after they were induced and then the samples were frozen, to be blotted on Monday.
Results: N/A
Next Steps: The samples will be ran on an SDS gel on Monday and blotted.
Week summary
faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. .
Aim of experiment: To western blot the LbpA culture samples from Friday.
Protocols Used: Western Blot
Results: Nothing was visible on the blot.
Next Steps: 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.
Aim of experiment: To check if the cells begin to die after induction with IPTG after having already grown to an OD600 of 0.5.
Protocols Used: Growth Curve Assay
Results:
Next Steps: The samples will be ran on an SDS gel on Monday and blotted.