Team:Bielefeld-Germany/Labjournal/week21

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

Monday September 17th

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.
  • Gradient-PCR with [http://partsregistry.org/Part:BBa_K863113 CBDclos(T7)+GFP_His]-template and a CBDclos_Freiburg- + GFP_Freiburg_compl-primer-mix.

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 CDBcex(T7)+GFP-plasmid and a CBDclos(T7)+GFP-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 J61101 and CBDclos with GFP_Freiburg and transformed it into KRX.

Thursday September 20th

• Team Cellulose Binding Domain:
  • No Colonies on the S3N10-Clos-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

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