Team:Bielefeld-Germany/Labjournal/week21

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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