Team:Penn/Notebook

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                     <a href="https://2013.igem.org/Team:Penn/Parts"><li>parts</li></a>
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                     <li><a href="https://2013.igem.org/Team:Penn/Modeling">Modeling</a></li>
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                     <a href="https://2013.igem.org/Team:Penn/Modeling"><li>modeling</li></a>
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                    <li><a href="https://2013.igem.org/Team:Penn/Safety">Safety</a></li>
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                    <a href="https://2013.igem.org/Team:Penn/Safety"><li>safety</li></a>
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                  <a href="https://2013.igem.org/Team:Penn/Team"> <li>team</li></a>
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                  <a href="https://2013.igem.org/Team:Penn/Sponsors"> <li>sponsors</li></a>
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                    <li><a href="https://igem.org/Team.cgi?id=1128">Official Team Profile</a></li>
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                    <a href="https://igem.org/Team.cgi?id=1128"><li>official team profile</li></a>
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                    <li><a href="#">One more separated link</a></li>-->
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<a href="https://2013.igem.org/Team:Penn/Notebook"><li>NOTEBOOK</li></a>
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    <div class="section-title" style=" margin-top: 150px; position: relative;">notebook</div>
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    <ul>
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        <li>week 1</li>
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        <li>week 2</li>
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        <li>week 3</li>
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        <li>week 4</li>
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        <li>week 5</li>
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    </ul>
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      <li>4-Jun
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        <h2>Week 1</h2>
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        <div class="box">
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        <h2>June 4 2013 - June 11 2013</h2>
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        <img src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/APrj2Se2i-/Lynch%20Labs%20PGFI/BE_LynchLabGroup_DSC2811.jpg?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg" class="image"/><li>4-Jun
             <ol>
             <ol>
             <li>Learned how to make competent cells, growing up two strains for tomorrow</li>
             <li>Learned how to make competent cells, growing up two strains for tomorrow</li>
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       </ol></li>
       </ol></li>
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       <br/>
         <li>5-Jun
         <li>5-Jun
       <ol>
       <ol>
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       </ol></li>
       </ol></li>
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       <br/>
       <li>6-Jun
       <li>6-Jun
             <ol>
             <ol>
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             <li>Wrote Penn iGEM on our plasmid</li>         
             <li>Wrote Penn iGEM on our plasmid</li>         
       </ol></li>
       </ol></li>
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       <br/>
           <li>7-Jun
           <li>7-Jun
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        <ol>
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      <ol>
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             <li>Transform
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             <li>Transform</li>
             <ol>
             <ol>
                 <li>C0012 –amp/chlor (do both)</li>
                 <li>C0012 –amp/chlor (do both)</li>
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                 <li>R0051 – amp</li>
                 <li>R0051 – amp</li>
                 <li>K206000 –chlor</li>
                 <li>K206000 –chlor</li>
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             </ol></li>
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             </ol>
             <li>Start the LIMS and file all the strains and DNA we have made/ ordered</li>
             <li>Start the LIMS and file all the strains and DNA we have made/ ordered</li>
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             <li>Mini-preped<ol>
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      </ol></li>
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      <br/>
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          <li>8-Jun</li> <ol>
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             <li>Miniprep Addgene stuff + transformations that worked</li>
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            <li>Growing up low copy plasmids in 40mLs</li>
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      </ol>          <li>Mini-prepped</li><ol>
                 <li>I9002</li>
                 <li>I9002</li>
                 <li>I13458</li>
                 <li>I13458</li>
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                 <li>Pdawn-mcherry</li>
                 <li>Pdawn-mcherry</li>
                 <li>Pdawn</li>
                 <li>Pdawn</li>
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                <li>Pet26b</li>
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                            <li>Dhsa mcherry</li>
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                 <li>Pdawn dhsa</li>
                 <li>Pdawn dhsa</li>
                 <li>Psb1a3</li>
                 <li>Psb1a3</li>
                 <li>JM mcherry</li>
                 <li>JM mcherry</li>
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                <li>Pet26b</li>
                  
                  
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        <h2>Week 2</h2>
          
          
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      </ol></li>
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            <h2>June 11 2013 - June 18 2013</h2>
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          <li>8-Jun <ol>
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        <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/-ZtRJ7Gu1U/photo-8.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
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            <li>Miniprep Addgene stuff + transformations that worked</li>
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             <li>17-Jun <ol>
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            <li>Growing up low copy plasmids in 40mLs</li>
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      </ol></li>
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        <li>8-Jun</li>
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      <li><ol>
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            <li>Miniprep Addgene stuff + transformations that worked</li>
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            <li>Growing up low copy plasmids in 40mLs</li>
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             <li>Transformed everything that has failed</li>
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      </ol></li>
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        <li>17-Jun <ol>
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             <li>Grow up luxI culture and grow up tetR culture </li>
             <li>Grow up luxI culture and grow up tetR culture </li>
             <li>Sequence all of the minipreps</li>
             <li>Sequence all of the minipreps</li>
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       </ol></li>
       </ol></li>
        
        
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       <br/>
       <li>18-Jun<ol>
       <li>18-Jun<ol>
             <li>The bisulfite conversion was missing a negative control (bisulfite converted but unmethylated c0051) we'll need this to interperet results</li>
             <li>The bisulfite conversion was missing a negative control (bisulfite converted but unmethylated c0051) we'll need this to interperet results</li>
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<li>Design a way to make variable promoters more easily varied (for a biobrick so teams can use the reporter plasmid for their own nefarious reasons)</li>
<li>Design a way to make variable promoters more easily varied (for a biobrick so teams can use the reporter plasmid for their own nefarious reasons)</li>
       </ol></li>
       </ol></li>
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       <li>19-Jun  <ol>
       <li>19-Jun  <ol>
             <li>Transform failed transformation</li>
             <li>Transform failed transformation</li>
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             <li>Check pTet-gfp under blue light</li>
             <li>Check pTet-gfp under blue light</li>
              
              
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       </ol></li>
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          <li>20-Jun<ol>
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            <li>run plux-luxI pcr</li>
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            <li>run pdawn-luxI pcr  </li>
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            <li>run pDawn-tetR pcr </li>
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        <h2>Week 3</h2>
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            <li>run pet26b-tetR pcr and</li>
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        <div class="box"> <h2>June 18 2013 - June 25 2013</h2>
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            <li>run pDawn-GFP pcr</li>
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        <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/Yn0SK0Y0-7/snap.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
-
            <li>run pDawn-mCherry-secretion tag pcr </li>
+
-
            <li>Nano drop last nightÕs mini preps to check for accuracy </li>
+
-
            <li>Culture amp resistant successful transformations </li>
+
-
            <li>Make 5 L LB </li>
+
-
            <li>Miniprep all the successful transformations w/ new protocol </li>
+
-
           
+
-
      </ol></li>
+
-
     
+
-
     
+
-
      <li>21-Jun<ol>
+
         <li>troubleshoot plux-luxI pcr</li>
         <li>troubleshoot plux-luxI pcr</li>
         <li>roubleshoot pdawn-luxI pcr</li>
         <li>roubleshoot pdawn-luxI pcr</li>
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       </ol></li>
       </ol></li>
        
        
 +
      <br/>
       <li>24-Jun<ol>
       <li>24-Jun<ol>
         <li>Dam-/dh5a v dam methylation + dpnI && dpnII digest</li>
         <li>Dam-/dh5a v dam methylation + dpnI && dpnII digest</li>
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         <li>figure out the primer issues 8</li>
         <li>figure out the primer issues 8</li>
         <li> Pick t9002 colonies for miniprep </li>
         <li> Pick t9002 colonies for miniprep </li>
-
       </ol></li>
+
       </ol></li></div>
-
     
+
    </div>
-
      <li>26-Jun<ol>
+
 
 +
   
 +
    <div>
 +
        <h2>Week 4</h2>
 +
        <div class="box"> <h2>June 25 2013 - July 2 2013</h2>
 +
        <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/1LlS0hsVIZ/Screen%20shot%202013-05-29%20at%2016.22.13.png?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
 +
<ul><li>26-Jun<ol>
         <li>Dam-/dh5a v dam methylation + dpnI && dpnII digest</li>
         <li>Dam-/dh5a v dam methylation + dpnI && dpnII digest</li>
         <li>Digested/ligated/transformed t9002 in amp </li>
         <li>Digested/ligated/transformed t9002 in amp </li>
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         <li>USER Cloning reporter plasmid </li>
         <li>USER Cloning reporter plasmid </li>
       </ol></li>
       </ol></li>
-
 
+
<br/>
-
       
+
-
     
+
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+
      
      
-
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         <li>1-Jul<ol>
         <li>1-Jul<ol>
             <li>Beautiful Brady Bunch photoshoot</li>
             <li>Beautiful Brady Bunch photoshoot</li>
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             <li>Get methylated biobrick sequenced </li>
             <li>Get methylated biobrick sequenced </li>
             <li>Only sequence ptet GFP 11, if verified make sure to note on LIMS that we are only using ptet GFP 11 </li>
             <li>Only sequence ptet GFP 11, if verified make sure to note on LIMS that we are only using ptet GFP 11 </li>
-
             <li> Check if plux/luxI system is working in liquid cultures – this failed <ol>
+
             <li> Check if plux/luxI system is working in liquid cultures – this failed</li> <ol style =""margin-left: 50px;"">
-
                 <li>a. Might be strain competition, need to know growth rates</li></li>
+
                 <li>a. Might be strain competition, need to know growth rates</li>
             </ol>
             </ol>
             <li>Re-suspend primers for lux amplifier </li>
             <li>Re-suspend primers for lux amplifier </li>
             <li>Mini-prep: e0040, psb1a3, r0062</li>     
             <li>Mini-prep: e0040, psb1a3, r0062</li>     
         </ol></li>
         </ol></li>
 +
        <br/>
          
          
-
       
+
         <li>2-Jul <ol><br>
-
         <li>2-Jul<ol>
+
             <li>Think about application of mathylation project in e.coli</li>
             <li>Think about application of mathylation project in e.coli</li>
             <li>Ceck if plux/GFP-psb1C3 system is working in liquid cultures
             <li>Ceck if plux/GFP-psb1C3 system is working in liquid cultures
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             <li>Growing up t9002 in chlor and i751250 in amp for fluorescence study</li>
             <li>Growing up t9002 in chlor and i751250 in amp for fluorescence study</li>
             <li>Investigate CHIP or other ways of determining DNA binding domain specificity </li>
             <li>Investigate CHIP or other ways of determining DNA binding domain specificity </li>
-
         </ol></li>
+
         </ol></li></ul></div><!--/box-->
-
          
+
    </div>
-
         <li>3-Jul<ol>
+
   
 +
    <div>
 +
         <h2>Week 5</h2>
 +
         <div class="box"> <h2>July 2 2013 - July 9 2013</h2>
 +
        <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/Y5d0PLu26o/photo-7.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
 +
            <ul><li>3-Jul<ol>
             <li>Streak zinc finger 2 </li>
             <li>Streak zinc finger 2 </li>
             <li>Grow up 44251 </li>
             <li>Grow up 44251 </li>
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         <li>6-Jul
         <li>6-Jul
-
        <ol>
+
      <ol>
             <li>troubleshoot ptetGFP user PCR - band was visible but too small to extract</li>
             <li>troubleshoot ptetGFP user PCR - band was visible but too small to extract</li>
             <li>gel extract promoter fragments from USER PCR</li>
             <li>gel extract promoter fragments from USER PCR</li>
             <li>re-do USER PCR for: TetR, pTetGFP</li>
             <li>re-do USER PCR for: TetR, pTetGFP</li>
             <li>Design/check/order (Tale,Cas,ZFP)-flex-REV primers - check fwd primers</li>
             <li>Design/check/order (Tale,Cas,ZFP)-flex-REV primers - check fwd primers</li>
-
         </ol></li>
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Revision as of 18:19, 22 August 2013

Notebook

notebook
  • week 1
  • week 2
  • week 3
  • week 4
  • week 5

Week 1

June 4 2013 - June 11 2013

  • 4-Jun
    1. Learned how to make competent cells, growing up two strains for tomorrow
    2. Transformed 8 plasmids
    3. Determined EL222 fusion is risky but still going ahead with it
    4. Linkers are totally setlled
    5. Found zinc finger plasmid and updated target sequence
    6. Learned how to make tetr- mcherry fusion
    7. Settled on 5 promoters

  • 5-Jun
    1. Learned how to make competent cells, testing them and then making more tomorrow
    2. Transformed 8 plasmids again
    3. Made primers to clone the TET-GFP reporter system, the mCherry promoter strength system, ready to order
    4. Made ultramers for variable promoter blocks (and no target neg controls) – ready to order
    5. Spilled a lot of iced tea outside, bummer
    6. Started primers for dna binding machines
    7. Got a handle on cas9 fusions (pun intended).
    8. Put awesome pics in dropbox

  • 6-Jun
    1. Clean up dropbox
    2. Update budget sheet with addgene and cell center orders
    3. Finish primers for fusion
    4. Set up plate reader for GFP and mCherry assays
    5. run minipreps on pdawn, pdawn-mcherry, pet26b
    6. Grow up mCherry stock
    7. Wrote Penn iGEM on our plasmid

  • 7-Jun
    1. Transform
      1. C0012 –amp/chlor (do both)
      2. M11307 – amp/chlor (do both)
      3. I13458 – amp/chlor (do both)
      4. R0010 – amp/chlor (do both)
      5. R0051 – amp
      6. K206000 –chlor
    2. Start the LIMS and file all the strains and DNA we have made/ ordered

  • 8-Jun
    1. Miniprep Addgene stuff + transformations that worked
    2. Growing up low copy plasmids in 40mLs
  • Mini-prepped
    1. I9002
    2. I13458
    3. C0051
    4. Pdawn-mcherry
    5. Pdawn
    6. Dhsa mcherry
    7. Pdawn dhsa
    8. Psb1a3
    9. JM mcherry
    10. Pet26b

    Week 2

    June 11 2013 - June 18 2013

  • 17-Jun
    1. Grow up luxI culture and grow up tetR culture
    2. Sequence all of the minipreps
    3. Transform t9002 in psb1A3 in NEB10
    4. Retransform ptetGFP to see if BL21DE3 cells are competent
    5. Transform r0079, k081015, r0063 in NEB10
    6. Miniprep psb1k3
    7. Redo dam gel with more dna
    8. Figure out second control zfp from addgene
    9. Figure out how to add luxR binding site to target region
    10. Order sequencing primers for all addgene minipreps
    11. Bisulfite converted msssi methylated c0051

  • 18-Jun
    1. The bisulfite conversion was missing a negative control (bisulfite converted but unmethylated c0051) we'll need this to interperet results
    2. Miniprep c0078, c0079 + make glycerol stocks check same plasmid with our kit
    3. Order 13420 (second zfp)
    4. Design a way to make variable promoters more easily varied (for a biobrick so teams can use the reporter plasmid for their own nefarious reasons)

  • 19-Jun
    1. Transform failed transformation
    2. Make competent DH5a && Dam-
    3. Figure out methylation assays for promoters
    4. Miniprep psb1A3 && all the 40mL cultures
    5. Picked many colonies
    6. Check pTet-gfp under blue light

    • Week 3

    June 18 2013 - June 25 2013

  • troubleshoot plux-luxI pcr
  • roubleshoot pdawn-luxI pcr
  • made pDawn-tetR pcr work
  • troubleshoot pet26b-tetR pcr
  • troubleshoot pDawn-GFP pcr
  • troubleshoot pDawn-mCherry-secretion tag pcr
  • miniprep growing cultures, be sure to pick only the glowing ligations
  • ransform the correct t9002 amp ligation - determined from gel
  • digested t9002 in amp and ptet gfp in amp to identify the correct ligation
  • all of the chosen ptet gfp ligations worked, but let's not use 5 || 13 12. troubleshoot t9002 digest
  • troubleshoot t9002 digest
    1. check for contamination of something (run uncut sample, sample + buffer, sample + 1 enzyme, sample + other enzyme, sample + both enzymes)

  • 24-Jun
    1. Dam-/dh5a v dam methylation + dpnI && dpnII digest
    2. Digested/ligated/transformed t9002 in amp
    3. Get methylated biobrick sequenced
    4. get chlor backbones sequenced
    5. culture t9002 transformations in liquid media with i751250
    6. mini prep stuff in the incubator
    7. figure out the primer issues 8
    8. Pick t9002 colonies for miniprep

    • Week 4

    June 25 2013 - July 2 2013

    • 26-Jun
      1. Dam-/dh5a v dam methylation + dpnI && dpnII digest
      2. Digested/ligated/transformed t9002 in amp
      3. get chlor backbones sequenced
      4. culture t9002 transformations in liquid media with i751250
      5. mini prep stuff in the incubator
      6. figure out the primer issues
      7. Pick t9002 colonies for miniprep
      8. USER Cloning reporter plasmid

    • 1-Jul
      1. Beautiful Brady Bunch photoshoot
      2. Troubleshooted and Re-tred PCR for user ends for reporter plasmid
      3. Called IDT about pcr assembly – they said Gibson tends to work better, no mutations, all in one tub. If we must PCR assembly – add DMSO, hotstart reaction, anneal at 68-70C (we did this).
      4. Get methylated biobrick sequenced
      5. Only sequence ptet GFP 11, if verified make sure to note on LIMS that we are only using ptet GFP 11
      6. Check if plux/luxI system is working in liquid cultures – this failed
        1. a. Might be strain competition, need to know growth rates
      7. Re-suspend primers for lux amplifier
      8. Mini-prep: e0040, psb1a3, r0062

    • 2-Jul

      1. Think about application of mathylation project in e.coli
      2. Ceck if plux/GFP-psb1C3 system is working in liquid cultures
        1. +/- AHL induction at 100nM
        2. Compare with ptetGFP fluorescence, normal LB fluorescence
      3. Streak zinc finger 2
      4. Grow up 44251
      5. Transform up R0062
      6. When BstuI arrives
        1. Assay BstuI working
        2. Assay the bvluc and tale1 minipreps in dh5a, dam-, and bl21 cells with msssi and bstuI
        3. Results: BstUI is blocked by methylation, and cells don’t normally methylate
      7. Growing up t9002 in chlor and i751250 in amp for fluorescence study
      8. Investigate CHIP or other ways of determining DNA binding domain specificity

    Week 5

    July 2 2013 - July 9 2013

    • 3-Jul
      1. Streak zinc finger 2
      2. Grow up 44251
      3. Look into lux box being light sensitive
    • 4-Jul
      1. Mini prep 44251
      2. Pick ZFP2 colonies to grow up, throw out liquid culture in fridge
    • 5-Jul
      1. Repeat BstUI assay, taking into account new controls
      2. Suspend the primers in the freezer
      3. We need to check if the origin of replications are compatible before co transformation
      4. Characterize pDawn-mcherry
      5. Practice measuring fluorescence
    • 6-Jul
      1. troubleshoot ptetGFP user PCR - band was visible but too small to extract
      2. gel extract promoter fragments from USER PCR
      3. re-do USER PCR for: TetR, pTetGFP
      4. Design/check/order (Tale,Cas,ZFP)-flex-REV primers - check fwd primers

    Retrieved from "http://2013.igem.org/Team:Penn/Notebook"