Difference between revisions of "Team:Northwestern/09 13"

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   <article>
 
   <article>
 
     <h1>Tuesday, September 13<sup>th</sup></h3>
 
     <h1>Tuesday, September 13<sup>th</sup></h3>
−
    <h2>Agenda:</h2>
+
<h2>Tasks:</h2>
−
 
+
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Jordan</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Worked on “Experiments” website content</li>
 +
          <li>Reread papers</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Michelle</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Nanodrop PCR purifications of GG parts:</li>
 +
          <ul>
 +
            <li>M1: 64.2 ng/uL, 260/680: 1.89, 260/230: 1.41</li>
 +
            <li>M2: 53.9 ng/uL, 260/280: 1.84, 260/230: 1.24</li>
 +
            <li>M3: 17.8 ng/uL, 260/280: 1.89, 260/230: 1.21</li>
 +
            <li>M4: 43.8 ng/uL, 260/280: 1.86, 260/230: 1.61</li>
 +
            <li>M5: 32.4 ng/uL, 260/280: 1.77, 260/230: 0.88</li>
 +
            <li>G1: 66.5 ng/uL, 260/280: 1.90, 260/230: 2.05</li>
 +
            <li>G2: 63.1 ng/uL, 260/280: 1.91, 260/230: 1.89</li>
 +
            <li>G3: 66.6 ng/uL, 260/280: 1.80, 260/230: 1.14</li>
 +
            <li>G4: 44.6 ng/uL, 260/280: 1.89, 260/230: 1.89</li>
 +
          </ul>
 +
          <li>Golden Gate dilutions:</li>
 +
          <ul>
 +
            <li>M1: 129.9 fmol/uL, 6.16 uL dilute to 20 uL (40 fmol/uL)</li>
 +
            <li>M2: 109.0 fmol/uL, 7.34 uL dilute to 20 uL (40 fmol/uL)</li>
 +
            <li>M3: 36.0 fmol/uL, 11.11 uL dilute to 20 uL (20 fmol/uL)</li>
 +
            <li>M4: 88.59 fmol/uL, 9.03 uL dilute to 20 uL (40 fmol/uL)</li>
 +
            <li>M5: 65.54 fmol/uL, 12.21 uL dilute to 20 uL (40 fmol/uL)</li>
 +
            <li>G1: 134.5 fmol/uL, 5.95 uL dilute to 20 uL (40 fmol/uL)</li>
 +
            <li>G2: 127.6 fmol/uL, 6.27 uL dilute to 20 uL (40 fmol/uL)</li>
 +
            <li>G3: 134.7 fmol/uL, 5.94 uL dilute to 20 uL (40 fmol/uL)</li>
 +
            <li>G4: 90.21 fmol/uL, 8.87 fmol/uL dilute to 20 uL (40 fmol/uL)</li>
 +
          </ul>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Paul</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>PCR purified GFP1-4, m1-5 (see Michelle’s notes)</li>
 +
          <li>Designed primers for inserting Cas9 into T7 expression vector pET28a</li>
 +
          <li>Talked with grads about T7 expression line/vector </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Sam</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Auburn Gresham groceries</li>
 +
          <li>Started Auburn Gresham presentation</li>
 +
          <li>To do: update OneNote </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Sara</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Miniprep of Cas9-TorA overnight culture
 +
          <div class="row">
 +
              <div class="col-xs-6 col-sm-7"><img src="https://static.igem.org/mediawiki/2016/7/7b/T--Northwestern--09_13_4.png" width="245" height="349" alt=""/></div>
 +
            </div>
 +
          </li>
 +
          <li>Emailed the UNSW team asking about whether they thought 100 kDa filters would work with their protocol</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Tasfia</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>PCR: Reran Tet-Lrz-GFP with same PCR conditions as 08.29.16</li>
 +
          <ul>
 +
            <li>Three 50-uL reactions using “Tet plasmid miniprep” (conc’n 450 ng/uL)</li>
 +
            <li>One reaction with 45 ng template</li>
 +
            <li>Two reactions with 4.5 ng template</li>
 +
          </ul>
 +
          <li>DpnI digested Tet-Lrz-GFP for one hour at 37°C on heat block</li>
 +
          <li>Ran gel screen on (non-DpnI digested) Tet-Lrz-GFP—faint bands that run at the correct size</li>
 +
          <ul>
 +
            <li>3 uL SybrSafe</li>
 +
            <li>6 uL 1kb ladder + 3 uL 6X purple loading dye</li>
 +
            <li>In each sample, 1 uL 6X purple loading dye + 5 uL DNA</li>
 +
            <div class="row">
 +
              <div class="col-xs-6"><img src="https://static.igem.org/mediawiki/2016/5/58/T--Northwestern--09_13_1.png" width="245" height="349" alt=""/></div>
 +
            </div>
 +
          </ul>
 +
          <li>PCR purified Tet-Lrz-GFP</li>
 +
          <ul>
 +
            <li>Gel Lanes 2+3: 15.2 ng/ul, 260/280: 1.89, 260/230: 1.17</li>
 +
            <li>Gel Lane 4: 13.3 ng/uL, 260/280: 1.83, 260/230: 1.17</li>
 +
          </ul>
 +
          <li>In progress: making Auburn Gresham presentation slides </li>
 +
          <li>PCR: Reran Tet-LRz-GFP</li>
 +
          <ul>
 +
            <li>Thermocycler 1 (right side); Q5 2X Master Mix</li>
 +
            <ul>
 +
              <li>Two 50-uL reactions using 500 nM primer concentration in reaction mix</li>
 +
              <ul>
 +
                <li>0.5 uL Tet plasmid miniprep template (8.0 ng)</li>
 +
                <li>2.5 uL 10 uM Tet_Lrz_GFP_FWD primer</li>
 +
                <li>2.5 uL 10 uM Tet_Lrz_GFP_REV primer</li>
 +
                <li>1 uL DMSO</li>
 +
                <li>18.5 uL nuclease-free water</li>
 +
                <li>25 uL Q5 HiFi 2X Master Mix</li>
 +
              </ul>
 +
              <li>Two 50-uL reactions using 200 nM primer concentration in reaction mix</li>
 +
              <ul>
 +
                <li>1 uL Tet plasmid miniprep template (8.0 ng, 0.8 ng)</li>
 +
                <li>1 uL 10 uM Tet_Lrz_GFP_FWD primer</li>
 +
                <li>1 uL 10 uM Tet_Lrz_GFP_REV primer</li>
 +
                <li>1 uL DMSO</li>
 +
                <li>21 uL nuclease-free water</li>
 +
                <li>25 uL Q5 HiFi 2X Master Mix</li>
 +
              </ul>
 +
            </ul>
 +
            <div class="row">
 +
              <div class="col-xs-6 col-sm-8"><img src="https://static.igem.org/mediawiki/2016/0/08/T--Northwestern--09_13_2.png" width="245" height="349" alt=""/></div>
 +
            </div>
 +
            <li>Thermocycler 2 (left side); OneTaq 2X Master Mix
 +
            </li>
 +
            <ul><li>Two 50-μL reactions using 200 nM primer concentration in reaction mix (as per usual) </li>
 +
            <ul><li>1 μL Tet plasmid miniprep template (8.0 ng, 0.8 ng)</li>
 +
            <li>1 μL 10 μM Tet_Lrz_GFP_FWD primer</li>
 +
            <li>1 μL 10 μM Tet_Lrz_GFP_REV primer</li>
 +
            <li>1 μL DMSO</li>
 +
            <li>21 μL nuclease-free water</li>
 +
            <li>25 μL OneTaq 2X Master Mix with Standard Buffer </li></ul></ul>
 +
            <div class="row">
 +
              <div class="col-xs-6 col-sm-8"><img src="https://static.igem.org/mediawiki/2016/e/e2/T--Northwestern--09_13_3.png" width="245" height="349" alt=""/></div>
 +
            </div>
 +
          </ul>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
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Latest revision as of 15:35, 18 October 2016

Notebook

Tuesday, September 13th

Tasks:

Jordan

  • Worked on “Experiments” website content
  • Reread papers

Michelle

  • Nanodrop PCR purifications of GG parts:
    • M1: 64.2 ng/uL, 260/680: 1.89, 260/230: 1.41
    • M2: 53.9 ng/uL, 260/280: 1.84, 260/230: 1.24
    • M3: 17.8 ng/uL, 260/280: 1.89, 260/230: 1.21
    • M4: 43.8 ng/uL, 260/280: 1.86, 260/230: 1.61
    • M5: 32.4 ng/uL, 260/280: 1.77, 260/230: 0.88
    • G1: 66.5 ng/uL, 260/280: 1.90, 260/230: 2.05
    • G2: 63.1 ng/uL, 260/280: 1.91, 260/230: 1.89
    • G3: 66.6 ng/uL, 260/280: 1.80, 260/230: 1.14
    • G4: 44.6 ng/uL, 260/280: 1.89, 260/230: 1.89
  • Golden Gate dilutions:
    • M1: 129.9 fmol/uL, 6.16 uL dilute to 20 uL (40 fmol/uL)
    • M2: 109.0 fmol/uL, 7.34 uL dilute to 20 uL (40 fmol/uL)
    • M3: 36.0 fmol/uL, 11.11 uL dilute to 20 uL (20 fmol/uL)
    • M4: 88.59 fmol/uL, 9.03 uL dilute to 20 uL (40 fmol/uL)
    • M5: 65.54 fmol/uL, 12.21 uL dilute to 20 uL (40 fmol/uL)
    • G1: 134.5 fmol/uL, 5.95 uL dilute to 20 uL (40 fmol/uL)
    • G2: 127.6 fmol/uL, 6.27 uL dilute to 20 uL (40 fmol/uL)
    • G3: 134.7 fmol/uL, 5.94 uL dilute to 20 uL (40 fmol/uL)
    • G4: 90.21 fmol/uL, 8.87 fmol/uL dilute to 20 uL (40 fmol/uL)

Paul

  • PCR purified GFP1-4, m1-5 (see Michelle’s notes)
  • Designed primers for inserting Cas9 into T7 expression vector pET28a
  • Talked with grads about T7 expression line/vector

Sam

  • Auburn Gresham groceries
  • Started Auburn Gresham presentation
  • To do: update OneNote

Sara

  • Miniprep of Cas9-TorA overnight culture
  • Emailed the UNSW team asking about whether they thought 100 kDa filters would work with their protocol

Tasfia

  • PCR: Reran Tet-Lrz-GFP with same PCR conditions as 08.29.16
    • Three 50-uL reactions using “Tet plasmid miniprep” (conc’n 450 ng/uL)
    • One reaction with 45 ng template
    • Two reactions with 4.5 ng template
  • DpnI digested Tet-Lrz-GFP for one hour at 37°C on heat block
  • Ran gel screen on (non-DpnI digested) Tet-Lrz-GFP—faint bands that run at the correct size
    • 3 uL SybrSafe
    • 6 uL 1kb ladder + 3 uL 6X purple loading dye
    • In each sample, 1 uL 6X purple loading dye + 5 uL DNA
  • PCR purified Tet-Lrz-GFP
    • Gel Lanes 2+3: 15.2 ng/ul, 260/280: 1.89, 260/230: 1.17
    • Gel Lane 4: 13.3 ng/uL, 260/280: 1.83, 260/230: 1.17
  • In progress: making Auburn Gresham presentation slides
  • PCR: Reran Tet-LRz-GFP
    • Thermocycler 1 (right side); Q5 2X Master Mix
      • Two 50-uL reactions using 500 nM primer concentration in reaction mix
        • 0.5 uL Tet plasmid miniprep template (8.0 ng)
        • 2.5 uL 10 uM Tet_Lrz_GFP_FWD primer
        • 2.5 uL 10 uM Tet_Lrz_GFP_REV primer
        • 1 uL DMSO
        • 18.5 uL nuclease-free water
        • 25 uL Q5 HiFi 2X Master Mix
      • Two 50-uL reactions using 200 nM primer concentration in reaction mix
        • 1 uL Tet plasmid miniprep template (8.0 ng, 0.8 ng)
        • 1 uL 10 uM Tet_Lrz_GFP_FWD primer
        • 1 uL 10 uM Tet_Lrz_GFP_REV primer
        • 1 uL DMSO
        • 21 uL nuclease-free water
        • 25 uL Q5 HiFi 2X Master Mix
    • Thermocycler 2 (left side); OneTaq 2X Master Mix
      • Two 50-μL reactions using 200 nM primer concentration in reaction mix (as per usual)
        • 1 μL Tet plasmid miniprep template (8.0 ng, 0.8 ng)
        • 1 μL 10 μM Tet_Lrz_GFP_FWD primer
        • 1 μL 10 μM Tet_Lrz_GFP_REV primer
        • 1 μL DMSO
        • 21 μL nuclease-free water
        • 25 μL OneTaq 2X Master Mix with Standard Buffer