Team:Bielefeld-Germany/Labjournal/week21

From 2012.igem.org

(Difference between revisions)
(Thursday September 20th)
(Monday September 17th)
Line 7: Line 7:
===Monday September 17th===
===Monday September 17th===
* '''Team Cultivation & Purification:'''
* '''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.
+
** 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===
===Tuesday September 18th===

Revision as of 18:36, 22 September 2012

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.
    • 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.

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.

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