|
|
| (71 intermediate revisions not shown) |
| Line 49: |
Line 49: |
| | | | |
| | ==Week 21 (09/17 - 09/23/12)== | | ==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 ''Dpn''I 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-''Dpn''I-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 ''Spe''I and ''Xba''I and ''Dpn''I.
| |
| - | **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.
| |
| - |
| |
| - |
| |
| - | {{Team:Bielefeld/Sponsoren}}
| |
"
