Team:Yale/Notebook/Week11

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''Friday''
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{{:Team:Yale/Templates/Yale_Header_Else}}
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- Planned/mapped out next 4 weeks
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<html>
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- Took dialyzed bag with purified eGFP-RiAFP and divided it into 12 eppendorfs, containing 1 mL each. Flash-froze tubes in liquid nitrogen, then quickly used a hot edge to poke holes into top of eppendorfs to allow sublimation. Tubes then placed in lyophilizer for 3 nights.
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<div id="text">
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      <div id="container">
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<div id="left-col">
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<ul id="nb">
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<li><a href="https://2011.igem.org/Team:Yale/Notebook/Week1">Week 1</a></li>
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<li><a href="https://2011.igem.org/Team:Yale/Notebook/Week2">Week 2</a></li>
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<li><a href="https://2011.igem.org/Team:Yale/Notebook/Week3">Week 3</a></li>
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<li><a href="https://2011.igem.org/Team:Yale/Notebook/Week4">Week 4</a></li>
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<li><a href="https://2011.igem.org/Team:Yale/Notebook/Week5">Week 5</a></li>
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<li><a href="https://2011.igem.org/Team:Yale/Notebook/Week6">Week 6</a></li>
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<li><a href="https://2011.igem.org/Team:Yale/Notebook/Week7">Week 7</a></li>
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<li><a href="https://2011.igem.org/Team:Yale/Notebook/Week8">Week 8</a></li>
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<li><a href="https://2011.igem.org/Team:Yale/Notebook/Week9">Week 9</a></li>
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<li><a href="https://2011.igem.org/Team:Yale/Notebook/Week10">Week 10</a></li>
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<li><a id="page" href="https://2011.igem.org/Team:Yale/Notebook/Week11">Week 11</a></li>
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<li><a href="https://2011.igem.org/Team:Yale/Notebook/Week12">Week 12</a></li>
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<li><a href="https://2011.igem.org/Team:Yale/Notebook/Week13">Week 13</a></li>
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<li><a href="https://2011.igem.org/Team:Yale/Notebook/Week14">Week 14</a></li>
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<li><a href="https://2011.igem.org/Team:Yale/Notebook/Week15">Week 15</a></li>
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</ul>
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</div>
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<div id="entry">
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''Saturday''
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<h1>Week 11: July 24-31, 2011</h1>
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- Began cloning all pSB1AK8 constructs into pSB1C3 for parts submission (G3, H1, RiA, RiG, T2, Z2?):
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<ul>
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- Digested 6 constructs and linearized pSB1C3 with EcoRI, PstI
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<li>Monday:<ul>
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- Also, PCR amplified eGFP segment from original eGFP-RiAFP plasmid to use as a control for survival assays
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<li>Regenerate Nickel columns</li>
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- Digested PCR fragment with eGFP with XbaI, PstI, and digested BBa_I712074 (strong T7 promoter) with SpeI and PstI
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<li>Incubate sample with beads</li>
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- Gel extracted all digested pSB1AK8 constructs using kit, gel visualized with SYBRSafe (digestion seemed successful)
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<li>Survival assay</li>
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</ul>
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Tuesday:<ul>
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<li>Survival Assay again</li>
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<li>Elution from loose beads + SDS</li>
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</ul></li><li>
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Wednesday:<ul>
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<li>Run flow-through through beads again</li>
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</ul></li><li>
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Thursday:<ul>
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<li>Researched ice-affinity purification</li>
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<li>Dialyzed sample</li>
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</ul></li><li>
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Friday:<ul>
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<li>Planned/mapped out next 4 weeks</li>
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<li>Took dialyzed bag with purified eGFP-RiAFP and divided it into 12 eppendorfs, containing 1 mL each.  Flash-froze tubes in liquid nitrogen, then quickly used a hot edge to poke holes into top of eppendorfs to allow sublimation.  Tubes then placed in lyophilizer for 3 nights.</li>
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</ul></li><li>
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Saturday:<ul>
 +
<li>Began cloning all pSB1AK8 constructs into pSB1C3 for parts submission (G3, H1, RiA, RiG, T2, Z2?) by digesting those 6 constructs and linearized pSB1C3 with EcoRI, PstI
 +
</li>
 +
<li>Also, PCR amplified eGFP segment from original eGFP-RiAFP plasmid to use as a control for survival assays</li>
 +
<li>Digested PCR fragment with eGFP with XbaI, PstI, and digested BBa_I712074 (strong T7 promoter) with SpeI and PstI</li>
 +
<li>Gel extracted all digested pSB1AK8 constructs using kit, gel visualized with SYBRSafe (digestion seemed successful)</li>
 +
</ul></li><li>
 +
Sunday:<ul>
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<li>Ligated gel-extracted pSB1AK8 fragments with pSB1C3; ligated eGFP PCR fragment with BBa_I712074</li>
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<li>Transformed ligations into DH5alpha cells, plated them</li>
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</ul></li>
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</ul>
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''Sunday''
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</div>
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- Ligated gel-extracted pSB1AK8 fragments with pSB1C3; ligated eGFP PCR fragment with BBa_I712074
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<div style="clear:both"></div>
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- Transformed ligations into DH5alpha cells, plated them
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</div>
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''Monday''
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- Ran colony PCR on transformed samples; seems to confirm successful ligation (see pics below):
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- Picked up lyophilized cells; resuspended first in 50 mL of 50 mM Tris-HCl (pH 7.5); A280 ~ .72, A488 ~ .13; however, looked cloudy (possible aggregation?), so centrifuged at 13K rpm for 10 minutes, and UV-vised the supernatant, A280 ~ .23, A488 ~ .04, suggesting around 75% was aggregated.  Tried again to resuspend in 100 mL Tris-HCl and adding 50 mM NaCl; still no good; tried adding acid and killed protein activity
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''Tuesday''
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- Miniprepped successful colony PCRs and submitted samples for sequencing
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- Prepared more buffers for protein purification
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- Transformed eGFP and eGFP-RiAFP and RiAFP cells
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''Wednesday''
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- Protein purification round 2!
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- Prepared a lysis buffer of 50 mM NaH2PO4, 300 mM NaCl (with 1 mM PMSF added before lysis), and 500 mM imidazole elution buffer (with same salts as lysis buffer)
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- Innoculated eGFP, eGFP-RiAFP, and RiAFP cells
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- Resuspended 1 L (500 mL) worth of eGFP-RiAFP (RiAFP) pellets in around 35 mL lysis buffer
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- Sonicated (using Spiegel lab sonicator) with 1 s on/4 s off for 25 total minutes of sonication for each resuspended pellet (unforunately, RiAFP sample by itself was foaming when we returned...)
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- Equilibrated 2 1 mL packed Ni-NTA columns (from Modis lab) with lysis buffer using peristaltic pump
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- Spin-concentrated samples using
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- Ran w FPLC/Fraction Collector Ni-NTA
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''Thursday''
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- Ran/checked gels with lots of protein
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- Grew up more cells
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- Size exclusion (small load) with eGFP-RiAFP and DTT'ed (after spin concentration)
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- Incubated with TEV
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''Friday''
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- Size exclusion of TEV-ed protein
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Latest revision as of 05:24, 28 September 2011

iGEM Yale

Week 11: July 24-31, 2011

  • Monday:
    • Regenerate Nickel columns
    • Incubate sample with beads
    • Survival assay
    Tuesday:
    • Survival Assay again
    • Elution from loose beads + SDS
  • Wednesday:
    • Run flow-through through beads again
  • Thursday:
    • Researched ice-affinity purification
    • Dialyzed sample
  • Friday:
    • Planned/mapped out next 4 weeks
    • Took dialyzed bag with purified eGFP-RiAFP and divided it into 12 eppendorfs, containing 1 mL each. Flash-froze tubes in liquid nitrogen, then quickly used a hot edge to poke holes into top of eppendorfs to allow sublimation. Tubes then placed in lyophilizer for 3 nights.
  • Saturday:
    • Began cloning all pSB1AK8 constructs into pSB1C3 for parts submission (G3, H1, RiA, RiG, T2, Z2?) by digesting those 6 constructs and linearized pSB1C3 with EcoRI, PstI
    • Also, PCR amplified eGFP segment from original eGFP-RiAFP plasmid to use as a control for survival assays
    • Digested PCR fragment with eGFP with XbaI, PstI, and digested BBa_I712074 (strong T7 promoter) with SpeI and PstI
    • Gel extracted all digested pSB1AK8 constructs using kit, gel visualized with SYBRSafe (digestion seemed successful)
  • Sunday:
    • Ligated gel-extracted pSB1AK8 fragments with pSB1C3; ligated eGFP PCR fragment with BBa_I712074
    • Transformed ligations into DH5alpha cells, plated them

Retrieved from "http://2011.igem.org/Team:Yale/Notebook/Week11"