Team:Cornell/testing/notebook/wetlab/2

From 2012.igem.org

(Difference between revisions)
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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.  
<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.  
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.  
+
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>)  
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>)  
  </br>
  </br>
-
<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.  
+
<br><b>July 2nd, Monday</b></br><br>
-
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.  
+
Today, Dylan and Caleb ran a <a href="http://2012.igem.org/wiki/images/6/6c/DNA_Gel_Electrophoresis.pdf">electrophoresis</a> of a <a href="http://2012.igem.org/wiki/images/3/33/Vent_PCR.pdf">Vent PCR</a> of p21 (the PCR product being the salicylate reporter) at 55 V. Additionally, Caleb decided to run a control gel at 100 V to determine whether higher voltage was a factor in our previous ladder problems (in addition to the SYBR Green stock). While we determined that the higher voltage did not cause our ladder issues, we did not see any bands from the p21 PCR (gel image not included).
-
That night, Steven ran a gel of the nahR digest while Spencer set up overnight cultures of p14k, p16k, pSB3C5, and pBBRBB. </br>
+
Dylan prepared electrocompetent cells using the <a href=" http://2012.igem.org/wiki/images/6/6f/MyersMyers_Shew-E-poration.pdf "> Myers and Myers </a> protocol. Modified protocol using two 5mL cultures @ 4000g for 10 min. Washed with 2 mL sorbitol, resuspended in 100 uL sorbitol. First electroporation ts = 2.32 ms, second ts = 2.02 ms. Added 1 uL of plasmid (575.5 ng) to each cuvette. First used .60 V, then 0.55 V, both with R = 400 ohms.  
 +
Mark and Danielle started liquid cultures of S1, S9, S10, S11, S15, S16, S18, S27 (See: <a href="http://2012.igem.org/Team:Cornell/testing/notebook/strainlist">Strain List</a>)
 +
</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.  
+
<br><b>July 3rd, Tuesday</b></br><br> Caleb <a href="http://2012.igem.org/wiki/images/3/35/Ezna_Miniprep.pdf">miniprepped </a>  S16 (p15), S22 (p21), S9 (p8), S10 (p9), S11 (p10), S18 (p17), and S15 (p14). (See: <a href="http://2012.igem.org/Team:Cornell/testing/notebook/strainlist">Strain List</a>
-
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.  
+
) Instead of a single EB elution, Caleb did two elutions, 30 ul each. The double 30 ul elution turned out to be effective at recovering a usable amount of DNA in the second elution, so we're including it on our miniprep protocol for future minipreps.
 +
The rerun of our Gibson sequencing failed again, so we tried a more roundabout method of determining whether our 3 Gibson transformants have complete nah operons or not. We digested them each with BamHI, expecting specific fragment lengths on a gel electrophoresis.
 +
When Dylan and Mark were attempting to determine what volume of ethidium bromide should be used in the gel, they came to the conclusion that better images may result from staining the gel in dilute ethidium bromide after running the gel, rather than including the stain in the gel. This experimental first staining used 200 mL of water conaining 20 uL of 10 mg/mL ethidium bromide, with gentle agitation for 1 hour. This stained the bands well, but also provided a large amount of background and took a very long time.
 +
We only saw one band on each, all of them at about 3.6kb.
 +
Because no transformations from our previous competent freezer stocks were successful, Dylan decided to make a starter culture of Shewanella strain JG 700 in preparation for making new competent stocks using the <a href="http://2012.igem.org/wiki/images/f/f7/PNNL_Electrocompetent_Shewanella.pdf
 +
"> PNNL Protocol
 +
</a>. Swati and Tina used this starter culture to complete the preparation.  
  </br>
  </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.
+
<br><b>July 4th, Wednesday</b></br><br>In the morning, Dylan, Swati, and Danielle prepared p8, p9, p10, p14, and p15 for <a href="http://2012.igem.org/wiki/images/0/03/Sequencing_Preparation.pdf "> sequencing </a> (from the minipreps that Caleb preformed the previous day).  
-
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.  
+
The team took the rest of the day off and had a barbeque at Buttermilk Falls. There was an excess of food, spontaneous song, and fireflies.   
-
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>
  </br>
 +
<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="http://2012.igem.org/wiki/images/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=" http://2012.igem.org/wiki/images/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 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="http://2012.igem.org/Team:Cornell/testing/notebook/strainlist">Strain List</a>)
 +
 +
</br>
 +
</div>
</div>

Revision as of 23:26, 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 PCR 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: Strain List)

    July 2nd, Monday

    Today, Dylan and Caleb ran a electrophoresis of a Vent PCR of p21 (the PCR product being the salicylate reporter) at 55 V. Additionally, Caleb decided to run a control gel at 100 V to determine whether higher voltage was a factor in our previous ladder problems (in addition to the SYBR Green stock). While we determined that the higher voltage did not cause our ladder issues, we did not see any bands from the p21 PCR (gel image not included). Dylan prepared electrocompetent cells using the Myers and Myers protocol. Modified protocol using two 5mL cultures @ 4000g for 10 min. Washed with 2 mL sorbitol, resuspended in 100 uL sorbitol. First electroporation ts = 2.32 ms, second ts = 2.02 ms. Added 1 uL of plasmid (575.5 ng) to each cuvette. First used .60 V, then 0.55 V, both with R = 400 ohms. Mark and Danielle started liquid cultures of S1, S9, S10, S11, S15, S16, S18, S27 (See: Strain List)

    July 3rd, Tuesday

    Caleb miniprepped S16 (p15), S22 (p21), S9 (p8), S10 (p9), S11 (p10), S18 (p17), and S15 (p14). (See: Strain List ) Instead of a single EB elution, Caleb did two elutions, 30 ul each. The double 30 ul elution turned out to be effective at recovering a usable amount of DNA in the second elution, so we're including it on our miniprep protocol for future minipreps. The rerun of our Gibson sequencing failed again, so we tried a more roundabout method of determining whether our 3 Gibson transformants have complete nah operons or not. We digested them each with BamHI, expecting specific fragment lengths on a gel electrophoresis. When Dylan and Mark were attempting to determine what volume of ethidium bromide should be used in the gel, they came to the conclusion that better images may result from staining the gel in dilute ethidium bromide after running the gel, rather than including the stain in the gel. This experimental first staining used 200 mL of water conaining 20 uL of 10 mg/mL ethidium bromide, with gentle agitation for 1 hour. This stained the bands well, but also provided a large amount of background and took a very long time. We only saw one band on each, all of them at about 3.6kb. Because no transformations from our previous competent freezer stocks were successful, Dylan decided to make a starter culture of Shewanella strain JG 700 in preparation for making new competent stocks using the PNNL Protocol . Swati and Tina used this starter culture to complete the preparation.

    July 4th, Wednesday

    In the morning, Dylan, Swati, and Danielle prepared p8, p9, p10, p14, and p15 for sequencing (from the minipreps that Caleb preformed the previous day). The team took the rest of the day off and had a barbeque at Buttermilk Falls. There was an excess of food, spontaneous song, and fireflies.

    July 5th, Thursday

    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 DNA gel electrophoresis, 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 Myers and Myers 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 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: Strain List)

    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.