Team:Cornell/testing/notebook/wetlab/2
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<br><b>July 5th, Thursday </b></br><br> Upon arriving to the lab, Dylan dropped off the sequencing tubes we'd set up on the morning of the 4th for analysis. He also decided to do a Colony PCR of the potential transformant with the salicylate reporter plasmid. However, when setting up this reaction, Dylan realized that we'd used the wrong sequencing primers (we'd used the standard VF2 BioBrick primer instead of a custom primer for the pBBRBB backbone). Consequently, Mark and Dylan resubmitted p14 and p15 for sequencing while the colony PCR was ongoing. After visualizing the PCR products using <a href="https://static.igem.org/mediawiki/2012/6/6c/DNA_Gel_Electrophoresis.pdf"> DNA gel electrophoresis</a>, Dylan concluded that the colony we'd screened did not have our plasmid of interest, and was the result of contamination. | <br><b>July 5th, Thursday </b></br><br> Upon arriving to the lab, Dylan dropped off the sequencing tubes we'd set up on the morning of the 4th for analysis. He also decided to do a Colony PCR of the potential transformant with the salicylate reporter plasmid. However, when setting up this reaction, Dylan realized that we'd used the wrong sequencing primers (we'd used the standard VF2 BioBrick primer instead of a custom primer for the pBBRBB backbone). Consequently, Mark and Dylan resubmitted p14 and p15 for sequencing while the colony PCR was ongoing. After visualizing the PCR products using <a href="https://static.igem.org/mediawiki/2012/6/6c/DNA_Gel_Electrophoresis.pdf"> DNA gel electrophoresis</a>, Dylan concluded that the colony we'd screened did not have our plasmid of interest, and was the result of contamination. | ||
Similarly, Dylan noticed small, evenly spaced colonies on all of the JG700 transformant plates from the <a href=" https://static.igem.org/mediawiki/2012/6/6f/MyersMyers_Shew-E-poration.pdf "> Myers and Myers </a> procedure we'd undertaken on the 2nd of July. Because none of the colonies looked red (as they should have if they were replicating pSB3C5), Dylan concluded that the chloramphenicol on the plates had degraded, and the colonies were resultant from untransformed cells. | Similarly, Dylan noticed small, evenly spaced colonies on all of the JG700 transformant plates from the <a href=" https://static.igem.org/mediawiki/2012/6/6f/MyersMyers_Shew-E-poration.pdf "> Myers and Myers </a> procedure we'd undertaken on the 2nd of July. Because none of the colonies looked red (as they should have if they were replicating pSB3C5), Dylan concluded that the chloramphenicol on the plates had degraded, and the colonies were resultant from untransformed cells. | ||
- | Because the colony PCR of the potential salicylate reporter didn't yield good results, Dylan and Youjin set up another Phusion PCR to amplify the salicylate-sensing region from p21. Simultaneously, Mark made SOB media for recovery after future transformations, as we'd just received the ingredients for the broth. | + | Because the colony PCR of the potential salicylate reporter didn't yield good results, Dylan and Youjin set up another <a href=" https://static.igem.org/mediawiki/2012/2/26/Phusion_PCR.pdf "> Phusion PCR |
- | Because we needed to submit for sequencing twice (because we used the wrong sequencing primers), Swati et al. set up liquid cultures of p21, p8, p9, p10, p17, and p14 in order to have more DNA in the freezer. We like having DNA in the freezer. (See: <a href="https://2012.igem.org/Team:Cornell/testing/notebook/strainlist">Strain List</a>) | + | </a> to amplify the salicylate-sensing region from p21. Simultaneously, Mark made SOB media for recovery after future transformations, as we'd just received the ingredients for the broth. |
+ | Because we needed to submit for sequencing twice (because we used the wrong sequencing primers), Swati et al. set up liquid cultures of p21, p8, p9, p10, p17, and p14 in order to have more DNA in the freezer. We like having DNA in the freezer. (See: <a href="https://2012.igem.org/Team:Cornell/testing/notebook/strainlist">Strain List</a>) </br> | ||
+ | <br><b>July 6th, Friday</b></br><br> Caleb <a href="https://static.igem.org/mediawiki/2012/3/35/Ezna_Miniprep.pdf">miniprepped </a> p21, p8, p9, p10, p17 from the liquid cultures set up the previous night. (See: <a href="https://2012.igem.org/Team:Cornell/testing/notebook/strainlist">Strain List</a> | ||
+ | ) We couldn't miniprep p14, since – after checking notes from the previous night – we learned that the culture was made with the wrong antibiotic (chloramphenicol instead of kanamycin). | ||
+ | Some sequencing results came back, however the Gibson results are still confusing. | ||
+ | Also, a <a href="https://static.igem.org/mediawiki/2012/6/6c/DNA_Gel_Electrophoresis.pdf"> gel</a> was run for the p21 PCR product. | ||
</br> | </br> | ||
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</div> | </div> | ||
</div> | </div> |
Revision as of 00:56, 4 October 2012
Wet Lab - July
-
Week 1
A lot of troubleshooting occurred this week. Once we figured out that SYBR Green was causing our gels to run strangely, we switched to staining with EtBr after running the gel. This approach gave us better results. We also continued to try electroporation protocols for transforming Shewanella, and continued to work on PCRing out the salicylate-sensing region. Daily DetailsWeek 2
It has been said that astronomy is a humbling and character-building experience. There is perhaps no better demonstration of the folly of human conceits than this distant image of our tiny world. To me, it underscores our responsibility to deal more kindly with one another, and to preserve and cherish the pale blue dot, the only home we've ever known. Daily DetailsWeek 3
It has been said that astronomy is a humbling and character-building experience. There is perhaps no better demonstration of the folly of human conceits than this distant image of our tiny world. To me, it underscores our responsibility to deal more kindly with one another, and to preserve and cherish the pale blue dot, the only home we've ever known. Daily DetailsWeek 4
It has been said that astronomy is a humbling and character-building experience. There is perhaps no better demonstration of the folly of human conceits than this distant image of our tiny world. To me, it underscores our responsibility to deal more kindly with one another, and to preserve and cherish the pale blue dot, the only home we've ever known. Daily Details -
Week 1
A lot of troubleshooting occurred this week. Once we figured out that SYBR Green was causing our gels to run strangely, we switched to staining with EtBr after running the gel. This approach gave us better results. We also continued to try electroporation protocols for transforming Shewanella, and continued to work on PCRing out the salicylate-sensing region.Week 2
Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.Week 3
Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.Week 4
Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. -
Week 1
A lot of troubleshooting occurred this week. Once we figured out that SYBR Green was causing our gels to run strangely, we switched to staining with EtBr after running the gel. This approach gave us better results. We also continued to try electroporation protocols for transforming Shewanella, and continued to work on PCRing out the salicylate-sensing region.Daily Details:
Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.Week 2
It has been said that astronomy is a humbling and character-building experience. There is perhaps no better demonstration of the folly of human conceits than this distant image of our tiny world. To me, it underscores our responsibility to deal more kindly with one another, and to preserve and cherish the pale blue dot, the only home we've ever known.Daily Details:
Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.Week 3
It has been said that astronomy is a humbling and character-building experience. There is perhaps no better demonstration of the folly of human conceits than this distant image of our tiny world. To me, it underscores our responsibility to deal more kindly with one another, and to preserve and cherish the pale blue dot, the only home we've ever known.Daily Details:
Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.Week 4
It has been said that astronomy is a humbling and character-building experience. There is perhaps no better demonstration of the folly of human conceits than this distant image of our tiny world. To me, it underscores our responsibility to deal more kindly with one another, and to preserve and cherish the pale blue dot, the only home we've ever known.Daily Details:
Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.