Difference between revisions of "Team:Northwestern/09 08"

(Created page with "<html> <style> </style> <head> <meta charset="UTF-8"> <meta http-equiv="X-UA-Compatible" content="IE=edge"> <meta name="viewport" content="width=device-width, initial-...")
 
 
(2 intermediate revisions by the same user not shown)
Line 20: Line 20:
 
   <article>
 
   <article>
 
     <h1>Thursday, September 8<sup>th</sup></h3>
 
     <h1>Thursday, September 8<sup>th</sup></h3>
−
     <h2>Agenda:</h2>
+
  <h2>Western Blot Results:</h2>
−
 
+
     <div class="row">
 +
      <div class="col-xs-12"><img src="https://static.igem.org/mediawiki/2016/5/51/T--Northwestern--09_08_1.png" width="754" height="580" alt=""/></div>
 +
    </div>
 +
<h2>Tasks:</h2>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Jordan</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Helped with Western blot in the morning</li>
 +
          <li>Looked into alternative periplasm fraction options</li>
 +
          <li>Transformed Golden Gates and Gibsons</li>
 +
          <ul><li>1 uL of Patrick’s transformation control on Amp</li>
 +
          <li>1 uL of Gibson YcdO product on Cam</li>
 +
          <li>2 uL “  “  “</li>
 +
          <li>3 uL “    “  “</li>
 +
          <li>4 uL “  “  “</li>
 +
          <li>3 ul of positive Golden Gate control from Patrick on Amp</li>
 +
          <li>3 ul GG control w/ no ligase on Amp</li>
 +
          <li>3 uL GG control w/ no insert on Amp</li>
 +
          <li>3 uL gRNA-guide GG on Tet</li>
 +
          <li>3 uL GFP-mcherry Golden Gate on Cam</li></ul>
 +
          <ul><li>Used 950 uL of SOC and plated 2 plates of each</li>
 +
          <li>Incubated for 1 hr. 15 minutes </li></ul>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Michelle</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Ran gel on Cas9-SS Gibsons (2)</li>
 +
          <ul>
 +
            <li>Completely empty</li>
 +
          </ul>
 +
          <li>Made 40 fmol/uL dilutions of gRNA template, mCherry1, and Tet lnrz for GFP for GG Golden Gate assembly</li>
 +
          <li>Ran Golden Gate reactions</li>
 +
          <ul>
 +
            <li>mCherry:</li>
 +
            <ul>
 +
              <li>1 uL of 40 fmol/uL mCherry1 PCR product</li>
 +
              <li>1 uL of 40 fmol/uL Tet lrnz for GFP PCR product</li>
 +
              <li>1 uL of 40 fmol/uL TorA</li>
 +
              <li>2 uL of 10X T4 ligase buffer</li>
 +
              <li>2.24 uL of 110uL BSA + 10uL T4 ligase buffer</li>
 +
              <li>0.5 uL of 400,000U/mL T4 ligase from NEB</li>
 +
              <li>0.5 uL of BsaI</li>
 +
              <li>Water to 21.76 uL</li>
 +
            </ul>
 +
            <li>gRNA:</li>
 +
            <ul>
 +
              <li>1 uL of 40 fmol/uL gRNA template</li>
 +
              <li>1 uL of annealed cutting guide insert from primers (straight from tube: ~ 6 uM)</li>
 +
              <li>2 uL of 10X T4 ligase buffer</li>
 +
              <li>2.24 uL of 110uL BSA + 10uL T4 ligase buffer</li>
 +
              <li>0.5 uL of 400,000U/mL T4 ligase from NEB</li>
 +
              <li>0.5 uL of BsaI</li>
 +
              <li>Water to 21.76 uL</li>
 +
            </ul>
 +
            <li>Positive control:</li>
 +
            <ul>
 +
              <li>1 uL of Patrick’s positive control “32, 50ng”</li>
 +
              <li>1 uL of Patrick’s positive control</li>
 +
              <li>2 uL of 10X T4 ligase buffer</li>
 +
              <li>2.24 uL of 110uL BSA + 10uL T4 ligase buffer</li>
 +
              <li>0.5 uL of 400,000U/mL T4 ligase from NEB</li>
 +
              <li>0.5 uL of BsaI</li>
 +
              <li>Water to 21.76 uL</li>
 +
            </ul>
 +
            <li>Negative control (no ligase):</li>
 +
            <ul>
 +
              <li>1 uL of Patrick’s positive control “32, 50ng”</li>
 +
              <li>1 uL of Patrick’s positive control</li>
 +
              <li>2 uL of 10X T4 ligase buffer</li>
 +
              <li>2.24 uL of 110uL BSA + 10uL T4 ligase buffer</li>
 +
              <li>0.5 uL of BsaI</li>
 +
              <li>Water to 21.76 uL</li>
 +
            </ul>
 +
            <li>Negative control (no insert):</li>
 +
            <ul>
 +
              <li>1 uL of Patrick’s positive control “32, 50ng”</li>
 +
              <li>2 uL of 10X T4 ligase buffer</li>
 +
              <li>2.24 uL of 110uL BSA + 10uL T4 ligase buffer</li>
 +
              <li>0.5 uL of 400,000U/mL T4 ligase from NEB</li>
 +
              <li>0.5 uL of BsaI</li>
 +
              <li>Water to 21.76 uL</li>
 +
            </ul>
 +
            <li>Conditions:</li>
 +
            <ul>
 +
              <li>30 cycles, 37&#176;C (2:00) 16&#176;C (2:00) 50&#176;C (5:00) 80&#176;C (5:00)</li>
 +
            </ul>
 +
          </ul>
 +
          <li>In the future add 2 uL of 10X BSA in every reaction tube</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Paul</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Finished Western Blot procedure with Ben</li>
 +
          <li>TetR plasmid expressed less Cas9 than Top10</li>
 +
          <ul>
 +
            <li>Fraction procedure may need revision:</li>
 +
            <ul>
 +
              <li>Slight Cas9 residue in periplasm fraction</li>
 +
            </ul>
 +
            <li>Lanes:</li>
 +
            <ul>
 +
              <li>1: ladder</li>
 +
              <li>2: Whole cell ClyA-GFP</li>
 +
              <li>3: Whole cell Neg control</li>
 +
              <li>4: Whole cell Cas9 in pSB1C3 (Tet promoter)</li>
 +
              <li>5: periplasm ClyA-GFP</li>
 +
              <li>6: periplasm Neg control</li>
 +
              <li>7: periplasm Cas9 in pSB1C3 (Tet promoter)</li>
 +
              <li>8: ladder</li>
 +
            </ul>
 +
          </ul>
 +
          <li>Phosphorylate and anneal gRNA primers:</li>
 +
          <ul>
 +
            <li>1 uL PNK</li>
 +
            <li>0.61 uL of 100 uM primer 1</li>
 +
            <li>0.61 uL of 100 uM primer 2</li>
 +
            <li>2 uL T4 ligase buffer</li>
 +
            <li>Water to 20 uL</li>
 +
            <li>@ 37&#176;C for 30 min @ 95&#176;C for 5 min</li>
 +
            <li>Cool to 28 C at ~1&#176;C/min</li>
 +
          </ul>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Sam</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Reached out to museums</li>
 +
          <li>Put finishing touches on pamphlet</li>
 +
          <li>Began writing human practices section for website</li>
 +
          <li>Began writing collaboration section for website</li>
 +
          <li>Emailed Australia </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Sara</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Read through the Australians’ protocols and made a list of questions and things that we would need to do them</li>
 +
          <li>Did a little lab notebook work</li>
 +
          <li>Plated the gibson Ycdo and Cas9 gibson products</li>
 +
          <ul>
 +
            <li>500 uL of the 1000 uL total solutions onto 2 plates each </li>
 +
          </ul>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Tyler</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Mathematical modeling, looked into and researched other antibiotic resistant targets</li>
 +
          <li>Gibson Reaction (Ycdo)</li>
 +
          <ul><li>2.78 n.f. H<sub>2</sub>0</li>
 +
          <li>0.22 µL Ycdo Insert</li>
 +
          <li>2 µL (36 ng) SS Lnrz Cas9</li>
 +
          <li>5 µL Gibson Mix </li></ul>
 +
</ul>
 +
      </div>
 +
    </div>
 +
  </article>
 
<footer id="nav">
 
<footer id="nav">
 
       <div class="row">
 
       <div class="row">
 
         <div class="col-sm-4"><a href="https://2016.igem.org/Team:Northwestern/09_07"><img src="https://static.igem.org/mediawiki/2016/c/c6/T--Northwestern--backarrow.png" height="15" width="15"/> yesterday</a></div>
 
         <div class="col-sm-4"><a href="https://2016.igem.org/Team:Northwestern/09_07"><img src="https://static.igem.org/mediawiki/2016/c/c6/T--Northwestern--backarrow.png" height="15" width="15"/> yesterday</a></div>
−
         <div class="col-sm-4"><a href="https://2016.igem.org/Team:Northwestern/Notebook">back to calender </a></div>
+
         <div class="col-sm-4"><a href="https://2016.igem.org/Team:Northwestern/Notebook">back to calendar </a></div>
 
         <div class="col-sm-4"><a href="https://2016.igem.org/Team:Northwestern/09_09">tomorrow <img src="https://static.igem.org/mediawiki/2016/6/65/T--Northwestern--forwardarrow.png" height="15" width="15"/></a></div>
 
         <div class="col-sm-4"><a href="https://2016.igem.org/Team:Northwestern/09_09">tomorrow <img src="https://static.igem.org/mediawiki/2016/6/65/T--Northwestern--forwardarrow.png" height="15" width="15"/></a></div>
 
       </div>
 
       </div>
 
     </footer>
 
     </footer>
−
  </article>
+
 
 
<script type="text/javascript" src="https://2016.igem.org/Team:Northwestern/libraries/jquery?action=raw&ctype=text/javascript"></script>
 
<script type="text/javascript" src="https://2016.igem.org/Team:Northwestern/libraries/jquery?action=raw&ctype=text/javascript"></script>
 
         <script type="text/javascript" src="https://2016.igem.org/Team:Northwestern/libraries/bootstrap?action=raw&ctype=text/javascript"></script>
 
         <script type="text/javascript" src="https://2016.igem.org/Team:Northwestern/libraries/bootstrap?action=raw&ctype=text/javascript"></script>

Latest revision as of 15:34, 18 October 2016

Notebook

Thursday, September 8th

Western Blot Results:

Tasks:

Jordan

  • Helped with Western blot in the morning
  • Looked into alternative periplasm fraction options
  • Transformed Golden Gates and Gibsons
    • 1 uL of Patrick’s transformation control on Amp
    • 1 uL of Gibson YcdO product on Cam
    • 2 uL “ “ “
    • 3 uL “ “ “
    • 4 uL “ “ “
    • 3 ul of positive Golden Gate control from Patrick on Amp
    • 3 ul GG control w/ no ligase on Amp
    • 3 uL GG control w/ no insert on Amp
    • 3 uL gRNA-guide GG on Tet
    • 3 uL GFP-mcherry Golden Gate on Cam
    • Used 950 uL of SOC and plated 2 plates of each
    • Incubated for 1 hr. 15 minutes

Michelle

  • Ran gel on Cas9-SS Gibsons (2)
    • Completely empty
  • Made 40 fmol/uL dilutions of gRNA template, mCherry1, and Tet lnrz for GFP for GG Golden Gate assembly
  • Ran Golden Gate reactions
    • mCherry:
      • 1 uL of 40 fmol/uL mCherry1 PCR product
      • 1 uL of 40 fmol/uL Tet lrnz for GFP PCR product
      • 1 uL of 40 fmol/uL TorA
      • 2 uL of 10X T4 ligase buffer
      • 2.24 uL of 110uL BSA + 10uL T4 ligase buffer
      • 0.5 uL of 400,000U/mL T4 ligase from NEB
      • 0.5 uL of BsaI
      • Water to 21.76 uL
    • gRNA:
      • 1 uL of 40 fmol/uL gRNA template
      • 1 uL of annealed cutting guide insert from primers (straight from tube: ~ 6 uM)
      • 2 uL of 10X T4 ligase buffer
      • 2.24 uL of 110uL BSA + 10uL T4 ligase buffer
      • 0.5 uL of 400,000U/mL T4 ligase from NEB
      • 0.5 uL of BsaI
      • Water to 21.76 uL
    • Positive control:
      • 1 uL of Patrick’s positive control “32, 50ng”
      • 1 uL of Patrick’s positive control
      • 2 uL of 10X T4 ligase buffer
      • 2.24 uL of 110uL BSA + 10uL T4 ligase buffer
      • 0.5 uL of 400,000U/mL T4 ligase from NEB
      • 0.5 uL of BsaI
      • Water to 21.76 uL
    • Negative control (no ligase):
      • 1 uL of Patrick’s positive control “32, 50ng”
      • 1 uL of Patrick’s positive control
      • 2 uL of 10X T4 ligase buffer
      • 2.24 uL of 110uL BSA + 10uL T4 ligase buffer
      • 0.5 uL of BsaI
      • Water to 21.76 uL
    • Negative control (no insert):
      • 1 uL of Patrick’s positive control “32, 50ng”
      • 2 uL of 10X T4 ligase buffer
      • 2.24 uL of 110uL BSA + 10uL T4 ligase buffer
      • 0.5 uL of 400,000U/mL T4 ligase from NEB
      • 0.5 uL of BsaI
      • Water to 21.76 uL
    • Conditions:
      • 30 cycles, 37°C (2:00) 16°C (2:00) 50°C (5:00) 80°C (5:00)
  • In the future add 2 uL of 10X BSA in every reaction tube

Paul

  • Finished Western Blot procedure with Ben
  • TetR plasmid expressed less Cas9 than Top10
    • Fraction procedure may need revision:
      • Slight Cas9 residue in periplasm fraction
    • Lanes:
      • 1: ladder
      • 2: Whole cell ClyA-GFP
      • 3: Whole cell Neg control
      • 4: Whole cell Cas9 in pSB1C3 (Tet promoter)
      • 5: periplasm ClyA-GFP
      • 6: periplasm Neg control
      • 7: periplasm Cas9 in pSB1C3 (Tet promoter)
      • 8: ladder
  • Phosphorylate and anneal gRNA primers:
    • 1 uL PNK
    • 0.61 uL of 100 uM primer 1
    • 0.61 uL of 100 uM primer 2
    • 2 uL T4 ligase buffer
    • Water to 20 uL
    • @ 37°C for 30 min @ 95°C for 5 min
    • Cool to 28 C at ~1°C/min

Sam

  • Reached out to museums
  • Put finishing touches on pamphlet
  • Began writing human practices section for website
  • Began writing collaboration section for website
  • Emailed Australia

Sara

  • Read through the Australians’ protocols and made a list of questions and things that we would need to do them
  • Did a little lab notebook work
  • Plated the gibson Ycdo and Cas9 gibson products
    • 500 uL of the 1000 uL total solutions onto 2 plates each

Tyler

  • Mathematical modeling, looked into and researched other antibiotic resistant targets
  • Gibson Reaction (Ycdo)
    • 2.78 n.f. H20
    • 0.22 µL Ycdo Insert
    • 2 µL (36 ng) SS Lnrz Cas9
    • 5 µL Gibson Mix