IGEM:Groningen/Notebook/iGEM 2011/2011/08/18

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18-8-11


colony PCR of PBAD-RBS-GFP-DT old and new, PhybB-taRNA-DT 1 and 4 and PhybB-RBS-GFP-DT in TOP10 cells:
Composition master mix:
10× Taq buffer: 84μl
dNTPs 10mM: 16.8μl
MgCl2: 50.4μl
Forward biobrickvector primer: 16.8μl
Reverse biobrickvector primer: 16.8μl
Taq polymerase: 4.2μl
MQ water: 651μl

PCR conditions:
Pre heated lid: 111°C
Denaturation: 94°C for 10min
Cycle (33×)
Denaturation: 94°C for 30s
Annealing: 60°C for 30s
Extenstion: 72°C for 2.5min
Final extension: 72°C for 10min
Store infinite at 4°C
Analyse PCR samples on a 1% agarosegel

PBAD-RBS-GFP-DT (with the new isolated vector) can possibly contain two right clones, colony 7 and 8 since the size is around
2000bp. Colonies 7 and 8 were grown on LB overnight.
Some of the PhybB-taRNA-DT 1 and 4 samples have the right fragment size. Colonies 1.1 and 4.1 were grown on LB overnight.
PhybB-RBS-GFP-DT in TOP 10 cells: all samples have the right fragment size. Colonies 2 and 3 were grown on LB overnight.


Dissolve new ordered primer Forward primer PBAD-araC 623bp
Prepare some samples for sequencing with this primer:
PBADaraC-cI-LVA-DT and PBADaraC-LasR-LVA-DT
Filling in forms for stage
Meeting at 14:00 and update meeting at 16:00


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