Team:Bielefeld-Germany/Labjournal/week21

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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/Starting"><strong>Prologue</strong></a></li>
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                <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week1"><strong>Week 1</strong></a></li>
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                <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week2"><strong>Week 2</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week3"><strong>Week 3</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week4"><strong>Week 4</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week5"><strong>Week 5</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week6"><strong>Week 6</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week7"><strong>Week 7</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week8"><strong>Week 8</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week9"><strong>Week 9</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week10"><strong>Week 10</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week11"><strong>Week 11</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week12"><strong>Week 12</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week13"><strong>Week 13</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week14"><strong>Week 14</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week15"><strong>Week 15</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week16"><strong>Week 16</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week17"><strong>Week 17</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week18"><strong>Week 18</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week19"><strong>Week 19</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week20"><strong>Week 20</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week21"><strong>Week 21</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week22"><strong>Week 22</strong></a></li>
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Revision as of 10:14, 23 September 2012


Contents

Week 21 (09/17 - 09/23/12)

Monday September 17th

  • Team Cultivation & Purification:
    • Cell disruption of fermentation of E. coli Rosetta-Gami 2 with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 BBa_K863012] via high-pressure homogenization and purification via Ni-NTA column.

Tuesday September 18th

  • Team Cellulose Binding Domain:
    • Primer arrived:
    • Gradient-PCR with the [http://partsregistry.org/Part:BBa_K863103 CBDcex(T7)+GFP_His]-plasmid and S3N10-CBDcex_RV and S3N10-GFP_FW primer-mix. the right product appeared only at 66,5°C; Lower: wrong product; Higher: no product
      • Following PCR at 66,5°C(50µL) had the wrong product (main product at 2 kbp); could be because of a different template concentration or not exact temperature.
      • Alternativly took the 20 µL-tube with right product and added 0,5 µL DpnI for over-night digestion.
    • Gradient-PCR with [http://partsregistry.org/Part:BBa_K863103 CBDcex(T7)+GFP_His]-template and a CBDcex_Freiburg- + GFP_Freiburg_compl-primer-mix to amplify the whole fusion-protein and get rid of the His-tag.
    • Gradient-PCR with [http://partsregistry.org/Part:BBa_K863113 CBDclos(T7)+GFP_His]-template and a CBDclos_Freiburg- + GFP_Freiburg_compl-primer-mix to amplify the whole fusion-protein and get rid of the His-tag.

Wednesday September 19th

  • Team Cellulose Binding Domain:
    • Stopped over-night-DpnI-digestion of S3N10-clos-GFP_his PCR-product and cleaned up in gel. Ligated the PCR-product with itself via Blunt-end-ligation and transformed it into KRX. Plated on Select-Agar.
    • Gradient-PCRs of the [http://partsregistry.org/Part:BBa_K863103 CBDcex(T7)+GFP_His]-plasmid and a [http://partsregistry.org/Part:BBa_K863113 CBDclos(T7)+GFP_His]-plasmid (unsequenced) with the CBDcex_Freiburg- and GFP_Freiburg-compl- and the CBDclos_Freiburg- and GFP_Freiburg_compl primers, respectively. The CBDcex-setup showed no correct product, but the CBDclos-setup had the right bands at temperatures from 57°C to 64°C; Merged the correct fractions and cleaned them up through the gel.
    • Digested the cleaned-up product with SpeI and XbaI and DpnI.
    • Ligated <partinfo>J61101</partinfo> and [http://partsregistry.org/Part:BBa_K863111 CBDclos_Freiburg] with [http://partsregistry.org/Part:BBa_K863120 GFP_Freiburg] and transformed it into KRX.

Thursday September 20th

  • Team Cellulose Binding Domain:
    • No Colonies on the S3N10-[http://partsregistry.org/Part:BBa_K863113 CBDclos(T7)+GFP_His]-transformations-dish with the blunt-end ligation; reason: forgotten to phosphorylate the PCR-product.
    • Two colonies on the CBDclos+GFP-dish with the constitutive [http://partsregistry.org/Part:BBa_J61101 J23100 + J61101] promoter. But none of them is glowing. And them shouldn't ... used the wrong selection-agar (since J61101 carries pSB1A2-backbone with AMP-resistence.

Friday September 21st

  • Team Cellulose Binding Domain:
    • Transformated <partinfo>J61101</partinfo> with CBDclos+GFP again and plated on the right selection-agar this time!

Saturday September 22nd

  • Team Cellulose Binding Domain:
    • Once again two colonies on the agar-dish: can not say if they have a green glow...
      • picked the colonies and them put into AMP-LB-Medium.
    • Also made a flask with 10 mL LM-Medium and Cells with the [http://partsregistry.org/Part:BBa_K863104 CBDcex(J61101)+GFP_His]-plasmid

Sunday September 23rd

  • Team Cellulose Binding Domain:
    • All cultures did not show any sign of GFP-glow under uv-light.


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