IGEM:Groningen/Notebook/iGEM 2011/2011/08/10

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10-8-11


colony PCR of PBAD-pSB1C3:
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 30s
Final extension: 72°C for 10min
Store infinite at 4°C
Analyse PCR samples on a 1% agarosegel

Plasmid prep of overnight culture PBAD-RBS-GFP colonies 5 and 6
Measure DNA concentration: 40 ng/microliter
Samples were send for sequencing and glycerol stocks were made

dsDNA arrived!!:)
structure in plasmid: SacI-prefix-taRNA-suffix-BamHI-prefix-crRNA-suffix-SalI-prefix-PRM-suffix-HindIII.
Dissolve the dsDNA in 50 microliter of MQ water, pipet 10 times up and down and incubate it for one hour at room temperature
Than make a dilution 100× and use 1 microliter for transformation:
Transformation dsDNA
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

Make overnight culture of the PBAD-pSB1C3 colonies 1 and 2

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