Difference between revisions of "Team:Dundee/lucylabjournal"
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+ | <div class="border-day"> | ||
+ | <div class="box"> | ||
+ | <span class="box-title">26/6: Plasmid Purification of the LbpA + pQE80-L Overnight Cultures</span> | ||
+ | <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew4-4"></span> | ||
+ | <div id="collapsiblew4-4" 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 LbpA gene.</p> | ||
+ | <p><b>Protocols Used: </b><a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol">Plasmid Purification</a></p> | ||
+ | <p><b>Results:</b> N/A </p> | ||
+ | <p><b>Next Steps:</b> </p> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
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2. For each colony, 5ml of fresh LB plus the appropriate antibiotics was inoculated and grown overnight at 37<sup>o</sup>C. | 2. For each colony, 5ml of fresh LB plus the appropriate antibiotics was inoculated and grown overnight at 37<sup>o</sup>C. | ||
+ | </p> | ||
+ | </div> | ||
+ | <div class="modal-footer"> | ||
+ | <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <!-- Miniprep Modal --> | ||
+ | <div class="modal fade" id="miniprep-modal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel"> | ||
+ | <div class="modal-dialog" role="document"> | ||
+ | <div class="modal-content"> | ||
+ | <div class="modal-header"> | ||
+ | <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">×</span></button> | ||
+ | <div class="modal-title" id="myModalLabel">Miniprep Protocol</div> | ||
+ | </div> | ||
+ | <div class="modal-body"> | ||
+ | <p> | ||
+ | <strong><u>Plasmid Purification (QIAprep<sup>®</sup> Spin Miniprep Kit)</u></strong> | ||
+ | <strong><u></u></strong> | ||
+ | </p> | ||
+ | <p> | ||
+ | 1. 1-5ml bacterial overnight culture was pelleted by centrifugation at >8000rpm for 3 minutes at room temperature. | ||
+ | </p> | ||
+ | <p> | ||
+ | 2. Pelleted bacterial cells were resuspended in 250µl of Buffer P1 and transferred to a microcentrifuge tube. | ||
+ | </p> | ||
+ | <p> | ||
+ | 3. 250µl of Buffer P2 was added and mixed thoroughly by inverting the tube 4-6 times until solution becomes clear. | ||
+ | </p> | ||
+ | <p> | ||
+ | 4. 350µl of Buffer N3 was added and immediately and thoroughly mixed by inverting the tube 4-6 times. | ||
+ | </p> | ||
+ | <p> | ||
+ | 5. The mixture was centrifuged for 10 minutes at 13000rpm in a microcentrifuge. | ||
+ | </p> | ||
+ | <p> | ||
+ | 6. The supernatant was applied to a QIAprep spin column by pipetting. | ||
+ | </p> | ||
+ | <p> | ||
+ | 7. The QIAprep spin column was washed with 500µl of Buffer PB by centrifugation for 1 minute and flow through discarded. | ||
+ | </p> | ||
+ | <p> | ||
+ | 8. 750µl of Buffer PE was added to the QIAprep spin column and centrifuged for 1 minute and flow through discarded. | ||
+ | </p> | ||
+ | <p> | ||
+ | 9. The QIAprep spin column was spun for an additional minute to remove residual wash buffer. | ||
+ | </p> | ||
+ | <p> | ||
+ | The QIAprep spin column was placed in a clean 1.5ml microcentrifuge and the DNA eluted by adding 50µl of H | ||
+ | </p> | ||
+ | <sub>2</sub> | ||
+ | <p> | ||
+ | O to the QIAprep spin column and centrifuging for 1 minute. | ||
</p> | </p> | ||
</div> | </div> |
Revision as of 20:44, 3 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 amplify LbpA for subsequent cloninng into the pQE80-L vector.
Protocols Used: PCR
Results:
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
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
Results: N/A
Next Steps:
The reactions were incubated at room temperature for at least 3 hours.