Team:Cornell/testing/notebook/wetlab/2

From 2012.igem.org

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<h6>Daily Details:</h6>
<h6>Daily Details:</h6>
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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.
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<br><b>July 1st, Sunday</b></br> <br> Dylan and Caleb set up two gels for <a href="http://2012.igem.org/wiki/images/6/6c/DNA_Gel_Electrophoresis.pdf">electrophoresis</a>. Caleb's was 1% agarose in BIO-RAD Mini-Sub Cell system for continuation of ladder test using SYBR Green, containing NEB 100 bp ladder, NEB 2-log ladder, Promega 1 kb ladder and run at 100V. Dylan's was 1% agarose in Owl box using ethidium bromide, containing the nah operon PCR product from previous night, and run at 55 V.  
 +
Our plates of DH5a transformed with p21 ligated into pBBRBB with mtrB grew only one colony, possibly contamination. Dylan ran a colony PCR and got a smear, suggesting that the PCR of p21 or the ligation did not work.
 +
Because we learned that our SYBR Green was causing ladder to run strangely, Dylan decided to redo a <a href="http://2012.igem.org/wiki/images/3/33/Vent_PCR.pdf">Vent PCR</a>to amplify the salicylate reporter region out of p21.
 +
Also, a liquid culture of JG700 was prepared, as well as replating of p21, p22, JG700, JG1220, JG1537, JG1219. (See: <a href="http://2012.igem.org/Team:Cornell/testing/notebook/strainlist">Strain List</a>)
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</br>
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<br><b>June 28th, Thursday</b></br><br>This morning, Dylan and Caleb did minipreps of all the cultures from the day before. They then set up a gel of Phusion PCR of nahR from Wednesday.
 +
That afternoon, Swati set up a transformation of p14k into DH5a cells. The cells were recovered for 1 hour at 37C in LB before plating. Dylan set up sequencing of the Gibson products with our newly designed primers while Shweta gel extracted the nahR from the gel set up by Dylan and Caleb. After Shweta’s gel extraction, Swati set up a double digestion of the product with EcoRI and AscI.
 +
That night, Steven ran a gel of the nahR digest while Spencer set up overnight cultures of p14k, p16k, pSB3C5, and pBBRBB. </br>
 +
 
 +
<br><b>June 29th, Friday</b></br><br>In the morning, Dylan set up a Vent PCR to amplify p21a (salicylate sensing region) and his previous Phusion PCR band. That afternoon, he gel purified the PCR products and set up a double digestion with EcoRI-HF and AscI. Spencer miniprepped the overnight cultures from Thursday.
 +
Later that night, Dylan set up another Phusion PCR to amplify the nah operon from Gibson colony 1 and to append Biobrick cutsites into the product for future ligation into pSB3C5. Swati started the gel extraction of p21a.
 +
</br>
 +
<br><b>June 30th, Saturday</b></br><br>Today, Dylan started out the day by running the PCR of the Gibson colony 1 on a gel, gel extracted the band, and set up another Phusion PCR with an extension time of 3 minutes to get more of the construct.
 +
That afternoon, Swati finished her p21a gel extraction and dephosphorylated pBBRBB+mtrB for ligation with p21a. Swati desalted the ligation on Millipore membrane paper and electroporated her ligation into DH5a. After letting the cells recover in LB for 1 hour, Swati plated her ligations.
 +
Later that night, Steven and Spencer set up two transformations into PNNL electrocompetent Shewanella one at a voltage of 0.75 kV, and the other at 0.55 kV prescribed by Myers and Myers.
 +
</br>
 +
 
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Revision as of 23:09, 3 October 2012

Weekly Update
Daily Details
Both

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 Details
    Daily Details:

    July 1st, Sunday

    Dylan and Caleb set up two gels for electrophoresis. Caleb's was 1% agarose in BIO-RAD Mini-Sub Cell system for continuation of ladder test using SYBR Green, containing NEB 100 bp ladder, NEB 2-log ladder, Promega 1 kb ladder and run at 100V. Dylan's was 1% agarose in Owl box using ethidium bromide, containing the nah operon PCR product from previous night, and run at 55 V. Our plates of DH5a transformed with p21 ligated into pBBRBB with mtrB grew only one colony, possibly contamination. Dylan ran a colony PCR and got a smear, suggesting that the PCR of p21 or the ligation did not work. Because we learned that our SYBR Green was causing ladder to run strangely, Dylan decided to redo a Vent PCRto amplify the salicylate reporter region out of p21. Also, a liquid culture of JG700 was prepared, as well as replating of p21, p22, JG700, JG1220, JG1537, JG1219. (See: Strain List)

    June 28th, Thursday

    This morning, Dylan and Caleb did minipreps of all the cultures from the day before. They then set up a gel of Phusion PCR of nahR from Wednesday. That afternoon, Swati set up a transformation of p14k into DH5a cells. The cells were recovered for 1 hour at 37C in LB before plating. Dylan set up sequencing of the Gibson products with our newly designed primers while Shweta gel extracted the nahR from the gel set up by Dylan and Caleb. After Shweta’s gel extraction, Swati set up a double digestion of the product with EcoRI and AscI. That night, Steven ran a gel of the nahR digest while Spencer set up overnight cultures of p14k, p16k, pSB3C5, and pBBRBB.

    June 29th, Friday

    In the morning, Dylan set up a Vent PCR to amplify p21a (salicylate sensing region) and his previous Phusion PCR band. That afternoon, he gel purified the PCR products and set up a double digestion with EcoRI-HF and AscI. Spencer miniprepped the overnight cultures from Thursday. Later that night, Dylan set up another Phusion PCR to amplify the nah operon from Gibson colony 1 and to append Biobrick cutsites into the product for future ligation into pSB3C5. Swati started the gel extraction of p21a.

    June 30th, Saturday

    Today, Dylan started out the day by running the PCR of the Gibson colony 1 on a gel, gel extracted the band, and set up another Phusion PCR with an extension time of 3 minutes to get more of the construct. That afternoon, Swati finished her p21a gel extraction and dephosphorylated pBBRBB+mtrB for ligation with p21a. Swati desalted the ligation on Millipore membrane paper and electroporated her ligation into DH5a. After letting the cells recover in LB for 1 hour, Swati plated her ligations. Later that night, Steven and Spencer set up two transformations into PNNL electrocompetent Shewanella one at a voltage of 0.75 kV, and the other at 0.55 kV prescribed by Myers and Myers.

    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
    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
    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
    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 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.