Team:Bielefeld-Germany/Labjournal/week21

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(Difference between revisions)
(Tuesday September 18th)
(Tuesday September 18th)
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***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.
***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 ''Dpn''I for over-night digestion.
***Alternativly took the 20 µL-tube with right product and added 0,5 µL ''Dpn''I for over-night digestion.
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**Gradient-PCR with [http://partsregistry.org/Part:BBa_K863103 CBDcex(T7)+GFP_His]-template and CBDcex_Freiburg+GFP_Freiburg-primer-mix.
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**Gradient-PCR with [http://partsregistry.org/Part:BBa_K863103 CBDcex(T7)+GFP_His]-template and a CBDcex_Freiburg+GFP_Freiburg-primer-mix.
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**Gradient-PCR with [http://partsregistry.org/Part:BBa_K863113 CBDclos(T7)+GFP_His]-template and CBDclos_Freiburg+GFP_Freiburg-primer-mix.
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**Gradient-PCR with [http://partsregistry.org/Part:BBa_K863113 CBDclos(T7)+GFP_His]-template and a CBDclos_Freiburg+GFP_Freiburg-primer-mix.
===Wednesday September 19th===
===Wednesday September 19th===

Revision as of 14:30, 22 September 2012

Contents

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