IGEM:Groningen/Notebook/iGEM 2011/2011/08/10: Difference between revisions

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==Entry title==
==Entry title==
* Insert content here...


<br> colony PCR of PBAD-pSB1C3:
<br> Composition master mix:
<br> 10× Taq buffer: 40μl
<br> dNTPs 10mM: 8μl
<br> MgCl2: 24μl
<br> Forward biobrickvector primer: 8μl
<br> Reverse biobrickvector primer: 8μl
<br> Taq polymerase: 2μl
<br> MQ water: 310μl
<br>
<br> PCR conditions:
<br> Pre heated lid: 111°C
<br> Denaturation: 94°C for 10min
<br> Cycle (33×)
<br>  Denaturation: 94°C for 30s
<br>  Annealing: 60°C for 30s
<br>  Extenstion: 72°C for 30s
<br> Final extension: 72°C for 10min
<br> Store infinite at 4°C
<br> Analyse PCR samples on a 1% agarosegel
<br>
<br> Plasmid prep of overnight culture PBAD-RBS-GFP colonies 5 and 6
<br> Measure DNA concentration: 40 ng/microliter
<br> Samples were send for sequencing and glycerol stocks were made
<br>
<br> dsDNA arrived!!:)
<br> structure in plasmid: SacI-prefix-taRNA-suffix-BamHI-prefix-crRNA-suffix-SalI-prefix-PRM-suffix-HindIII.
<br> Dissolve the dsDNA in 50 microliter of MQ water, pipet 10 times up and down and incubate it for one hour at room temperature
<br> Than make a dilution 100× and use 1 microliter for transformation:
<br> Transformation dsDNA
<br> Add to 40μl competent cells 10μl ligation mixture
<br> Incubate for 30 min on ice
<br> Heat shock: 45s at 42 degrees
<br> Incubate cells on ice for 2 min
<br> Add 1ml LB medium+ 25mM glucose
<br> Incubate cells for 1h/1.5h at 37 degrees
<br> Spin the cells down, resuspend them in 100μl LB medium and plate 90μl and 10μl out on the plates
<br> Put the plates in the stove at 37 degrees overnight
<br>
<br>


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Revision as of 08:15, 11 August 2011

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Entry title


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

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