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Team:Bielefeld-Germany/Labjournal/week21
From 2012.igem.org
Contents |
Week 21 (09/17 - 09/23/12)
Monday September 17th
Tuesday September 18th
- Team Cellulose Binding Domain:
- Primer arrived:
- Gradient-PCR with the 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 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 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 CBDcex(T7)+GFP_His-plasmid and a 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 CBDclos_Freiburg 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 J23100 + J61101 promoter. But none of them is glowing.
Friday September 21st
Saturday September 22nd
Sunday September 23rd
Sunday
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