Team:Bielefeld-Germany/Labjournal/week21

From 2012.igem.org

Revision as of 14:37, 22 September 2012 by Mo (Talk | contribs)

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.
    • A few colonies on the dish with the CBDclos+GFP and the constitutive [http://partsregistry.org/Part:BBa_J61101 J23100 + J61101] promoter. But none of them is glowing.

Friday September 21st

Saturday September 22nd

Sunday September 23rd

Sunday


55px Logo merck.jpg BioCircle.JPG Bielefeld2012 Evonik.jpg Bielefeld2012 Baxter.png Logo knauer.jpg Logo iit.jpg Bielefeld2012 BIEKUBA.jpg Logo biometra.jpg Logo bio-nrw.png Bielefeld2012 Logo ERASynbio.jpg