Team:UT Dallas/NotebookWeek6

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         <li class = "active"><a href="https://2011.igem.org/Team:UT_Dallas/Notebook"><font size="3" face="verdana">Notebook</a></font></li>
         <li class = "active"><a href="https://2011.igem.org/Team:UT_Dallas/Notebook"><font size="3" face="verdana">Notebook</a></font></li>
         <li><a href="https://2011.igem.org/Team:UT_Dallas/HumanPractices"><font size="3" face="verdana">Human Practices</a></font></li>
         <li><a href="https://2011.igem.org/Team:UT_Dallas/HumanPractices"><font size="3" face="verdana">Human Practices</a></font></li>
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         <li><a href="https://2011.igem.org/Team:UT_Dallas/Gallery"><font size="3" face="verdana">Gallery</a></font></li>
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         <li><a href="https://2011.igem.org/Team:UT_Dallas/Safety"><font size="3" face="verdana">Safety</a></font></li>
          
          
       </ul>
       </ul>
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           <h2><span> Week 6</span></h2>
           <h2><span> Week 6</span></h2>
           <p><font size = "5" face= "arial" color = "#184D79"><b>August 15-</b></font>
           <p><font size = "5" face= "arial" color = "#184D79"><b>August 15-</b></font>
-
               <blockquote><li type = "circle">PCR of ptetR, RBS, LuxI, term<li type = "circle">Gel electrophoresis and purification (insert 2 pictures here)<li type = "circle">Glycerol stock and miniprep <li type = "circle">Incubation in 3 mL LB can overnight at 37 degrees Celsius and 220 rpm<li type = "circle">Test digestion  for 1 hour and 30 minutes at 37 degrees Celsius<li type = "circle">Gel Electrophoresis<li type = "circle">Test Digestion Observation:  Lane 5 gave positive results for FGF-R: 2 bands observed at predicted bands size. Lane 4, 7, 9 were all negative bad results because EcoRI was used but only 1 band showed. This suggests that FGFR was not inserted. Lanes 10, 11, 12 were CheZ* and all matched negative control. Results are bad for CheZ*- will be regrown/miniprep/ligated.<li type = "circle">Incubation in 3 mL LB chlora overnight at 37 degrees Celsius and 220 rpm
+
               <blockquote><li type = "circle">PCR of ptetR, RBS, LuxI, term<li type = "circle">Gel electrophoresis and purification<li type = "circle">Glycerol stock and miniprep <li type = "circle">Incubation in 3 mL LB can overnight at 37 degrees Celsius and 220 rpm<li type = "circle">Test digestion  for 1 hour and 30 minutes at 37 degrees Celsius<li type = "circle">Gel Electrophoresis<li type = "circle">Test Digestion Observation:  Lane 5 gave positive results for FGF-R: 2 bands observed at predicted bands size. Lane 4, 7, 9 were all negative bad results because EcoRI was used but only 1 band showed. This suggests that FGFR was not inserted. Lanes 10, 11, 12 were CheZ* and all matched negative control. Results are bad for CheZ*- will be regrown/miniprep/ligated.<li type = "circle">Incubation in 3 mL LB chlora overnight at 37 degrees Celsius and 220 rpm
</blockquote>
</blockquote>
<p><font size = "5" face= "arial" color = "#184D79"><b>August 16-</b></font>
<p><font size = "5" face= "arial" color = "#184D79"><b>August 16-</b></font>
-
               <blockquote><li type = "circle"><li type = "circle"><li type = "circle"><li type = "circle"><li type = "circle"><li type = "circle"><li type = "circle"><li type = "circle"><li type = "circle"><li type = "circle"></blockquote>
+
               <blockquote><li type = "circle">Digestion; Gel Electrophoresis and Purification<li type = "circle">Ligation (10 minutes at room temperature)<li type = "circle">Transformation (50 µL DH5α, 2 µL DNA, 37 degrees Celsius overnight, all carb)<li type = "circle">Re- incubating – taking 20 µL of yesterday’s incubations and placing them in 3 mL of LB and appropriate<li type = "circle">Transformation (50 µL DH5α, 2 µL DNA, 37 degrees Celsius overnight, all carb)</blockquote>
<p><font size = "5" face= "arial" color = "#184D79"><b>August 17-</b></font>
<p><font size = "5" face= "arial" color = "#184D79"><b>August 17-</b></font>
-
               <blockquote><li type = "circle">Digestion: 1 hour and 30 minutes at 37 degrees Celsius<li type = "circle">Plating agar stab of ToxR<li type = "circle">Dephosphorylation for 1 hour at 37 degrees Celsius<li type = "circle">PCR purify<li type = "circle">Ligation- incubated at 16 degrees Celsius overnight<li type = "circle">Summary: Digestion of positive result sample of previous FGFR biobrick was performed with EcoRI to verify if true/false positive results. In parallel, a digestion of Alfredo’s phototaxis receptor and eYFP was performed using Alfredo’s backbone XbaI and SpeI. This was done to take it out of the native back bone and eventually ligate into new vector backbone (pSBIC3). First the part was dephosphorylated and then ligated into new vector. At previous FGFR and CheZ* (digested parts) were also ligated into the vector backbone pSBIC3 in parallel procedure (will incubate overnight). ToxR, which did not grow previously, was re-inoculated and incubating overnight.</blockquote>
+
               <blockquote><li type = "circle">Glycerol stock ToxR and CheZ* colonies (2nd attempt)<li type = "circle">Miniprep<li type = "circle">no results on gel<li type = "circle">Incubate in 3 mL LB carb overnight at 37 degrees Celsius and 220 rpm<li type = "circle">Transformation (50 µL DH5α, 2 µL DNA, 37 degrees Celsius overnight, all carb)<li type = "circle">Incubation in 3 mL of LB chlora overnight at 37 degrees Celsius and 220 rpm</blockquote>
 +
 
<p><font size = "5" face= "arial" color = "#184D79"><b>August 18-</b></font>
<p><font size = "5" face= "arial" color = "#184D79"><b>August 18-</b></font>
-
              <blockquote><li type = "circle">Digestion: 1 hour and 30 minutes at 37 degrees Celsius<li type = "circle">Plating agar stab of ToxR<li type = "circle">Dephosphorylation for 1 hour at 37 degrees Celsius<li type = "circle">PCR purify<li type = "circle">Ligation- incubated at 16 degrees Celsius overnight<li type = "circle">Summary: Digestion of positive result sample of previous FGFR biobrick was performed with EcoRI to verify if true/false positive results. In parallel, a digestion of Alfredo’s phototaxis receptor and eYFP was performed using Alfredo’s backbone XbaI and SpeI. This was done to take it out of the native back bone and eventually ligate into new vector backbone (pSBIC3). First the part was dephosphorylated and then ligated into new vector. At previous FGFR and CheZ* (digested parts) were also ligated into the vector backbone pSBIC3 in parallel procedure (will incubate overnight). ToxR, which did not grow previously, was re-inoculated and incubating overnight.</blockquote>
+
            <blockquote><li type = "circle">Glycerol Stock- 350 µL cells and 150 µL 50% glycerol<li type = "circle">Miniprep<li type = "circle">Test digestion<li type = "circle">Digestion, gel electrophoresis and purification<li type = "circle">Glycerol stock colonies 4 and 5 CheZ*<li type = "circle">Miniprep<li type = "circle">Test digestion and gel electrophoresis<li type = "circle">Ligation at 16 degrees Celsius overnight<li type = "circle">Incubation in 3 mL LB carb overnight at 37 degrees Celsius and 220 rpm<li type = "circle">Plate (507011) from Agar Stab</blockquote>
 +
 
           <div class="clr"></div>
           <div class="clr"></div>

Latest revision as of 02:14, 3 September 2011

biz solution

Week 6

August 15-

  • PCR of ptetR, RBS, LuxI, term
  • Gel electrophoresis and purification
  • Glycerol stock and miniprep
  • Incubation in 3 mL LB can overnight at 37 degrees Celsius and 220 rpm
  • Test digestion for 1 hour and 30 minutes at 37 degrees Celsius
  • Gel Electrophoresis
  • Test Digestion Observation: Lane 5 gave positive results for FGF-R: 2 bands observed at predicted bands size. Lane 4, 7, 9 were all negative bad results because EcoRI was used but only 1 band showed. This suggests that FGFR was not inserted. Lanes 10, 11, 12 were CheZ* and all matched negative control. Results are bad for CheZ*- will be regrown/miniprep/ligated.
  • Incubation in 3 mL LB chlora overnight at 37 degrees Celsius and 220 rpm

    • August 16-

  • Digestion; Gel Electrophoresis and Purification
  • Ligation (10 minutes at room temperature)
  • Transformation (50 µL DH5α, 2 µL DNA, 37 degrees Celsius overnight, all carb)
  • Re- incubating – taking 20 µL of yesterday’s incubations and placing them in 3 mL of LB and appropriate
  • Transformation (50 µL DH5α, 2 µL DNA, 37 degrees Celsius overnight, all carb)

    • August 17-

  • Glycerol stock ToxR and CheZ* colonies (2nd attempt)
  • Miniprep
  • no results on gel
  • Incubate in 3 mL LB carb overnight at 37 degrees Celsius and 220 rpm
  • Transformation (50 µL DH5α, 2 µL DNA, 37 degrees Celsius overnight, all carb)
  • Incubation in 3 mL of LB chlora overnight at 37 degrees Celsius and 220 rpm

    • August 18-

  • Glycerol Stock- 350 µL cells and 150 µL 50% glycerol
  • Miniprep
  • Test digestion
  • Digestion, gel electrophoresis and purification
  • Glycerol stock colonies 4 and 5 CheZ*
  • Miniprep
  • Test digestion and gel electrophoresis
  • Ligation at 16 degrees Celsius overnight
  • Incubation in 3 mL LB carb overnight at 37 degrees Celsius and 220 rpm
  • Plate (507011) from Agar Stab

    • Weeks 1-12

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