IGEM:Groningen/Notebook/iGEM 2011/2011/08/24

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24-8-11




colony PCR of PhybB-RBS-cI-LVA and PhybB-RBS-LasR-LVA:
Composition master mix:
10× Taq buffer: 40μl
dNTPs 10mM: 8μl
MgCl2: 24μl
Forward biobrickvector primer: 8μl
Reverse biobrickvector primer: 8μl
Taq polymerase: 2μl
MQ water: 310μl

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 2min
Final extension: 72°C for 10min
Store infinite at 4°C
Analyse PCR samples on a 1% agarosegel

Transformation of overnight ligation of PBAD-RBS-cI-LVA and PBAD-RBS-LasR-LVA
Add to 40μl competent cells 10μl ligation mixture
Incubate for 30 min on ice
Heat shock: 45s at 42 degrees
Incubate cells on ice for 2 min
Add 1ml LB medium+ 25mM glucose
Incubate cells for 1h/1.5h at 37 degrees
Spin the cells down, resuspend them in 100μl LB medium and plate 90μl and 10μl out on the plates
Put the plates in the stove at 37 degrees overnight

Made overnight cultures of PhybB-taRNA and PBAD-RBS-GFP for new samples to send for sequencing, since the sequencing results
themselves are shitty, low Q16 and Q20 values and the plot looks shitty. In one peak there is sometimes another smaller peak,
So the primersets and/or the samples could be contaminated


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