IGEM:Groningen/Notebook/iGEM 2011/2011/08/30

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30-8-11



Colony PCR of PBAD-RBS-GFP-DT

Taq 10× buffer: 20μl
dNTPs 10mM: 4μl
MgCl2: 12μl
Forward Biobrick primer: 4μl
Reverse Biobrick primer: 4μl
Taq DNA polymerase: 1μl
MQ water: 155μl

PCR conditions:
Preheated lid: 111°C
Denaturation: 94°C for 10 min.
Cycle 33×:
denaturation: 94°C for 30s.
annealing: 60°C for 30s.
Extension: 72°C for 2,5 min.
Final extension: 72°C for 10 min.
Store infinite at 4°C

Analyse on a 1% TBE agarose gel.
Transformants seem to contain PBADaraC, but not RBS-GFP-DT since the fragment has a size of 1500 a 1600 bp.

Overnight cultures PhybB-cI-LVA-DT colonies 2 and 3 and PhybB-LasR-LVA- colonies 2 and 6(with RBSes xD)grew.
Plasmid prep was done.
ND1000- Nanodrop measurements results: DNA concentrations were 30-40 ng/μl
Samples were send for sequencing and glycerol stocks were made
Measurements with FACS were done for PhybB-RBS-GFP, Olesja continues. Also PhybB-RBS-GFP-DT in E.coli BW strain

Protocol obtained again for gDNA isolation with magnetic beads for report

New cloning strategy for PBAD-cI-LVA-DT and PBAD-LasR-LVA-DT



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