IGEM:Groningen/Notebook/iGEM 2011/2011/06/24

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(Autocreate 2011/06/24 Entry for IGEM:Groningen/Notebook/iGEM_2011)
Current revision (06:39, 21 September 2011) (view source)
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==Entry title==
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==24-6-11==
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* Insert content here...
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<br>
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<br> Purify the PCR product with the High Pure PCR Purification kit from Roche
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<br> PhybB-RBS-GFP-DT
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<br>
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<br> Digestion mix:
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<br> 18μl buffer
 +
<br> 4.5μl EcoRI
 +
<br> 4.5μl SpeI
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<br> 49μl MQ water
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<br>
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<br> Vector mix:
 +
<br> 18μl buffer
 +
<br> 4.5μl EcoRI
 +
<br> 4.5μl XbaI
 +
<br> 49μl MQ water
 +
<br>
 +
<br> Ligation:
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<br> 2μl T4 DNA ligase buffer
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<br> 1μl T4 DNA ligase
 +
<br> 7μl insert
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<br> 3μl vector
 +
<br> 7μl MQ water
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<br>
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<br> Ligate 2 hours at room temperature
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<br>
 +
<br> Transformation
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<br> Add to 40μl competent cells 10μl ligation mixture
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<br> Incubate for 30 min on ice
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<br> Heat shock: 45s at 42 degrees
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<br> Incubate cells on ice for 2 min
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<br> Add 1ml LB medium+ 25mM glucose
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<br> Incubate cells for 1h/1.5h at 37 degrees
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<br> Spin the cells down, resuspend them in 100μl LB medium and plate 90μl and 10μl out on the plates
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<br> Put the plates in the stove at 37 degrees overnight
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24-6-11



Purify the PCR product with the High Pure PCR Purification kit from Roche
PhybB-RBS-GFP-DT

Digestion mix:
18μl buffer
4.5μl EcoRI
4.5μl SpeI
49μl MQ water

Vector mix:
18μl buffer
4.5μl EcoRI
4.5μl XbaI
49μl MQ water

Ligation:
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
7μl insert
3μl vector
7μl MQ water

Ligate 2 hours at room temperature

Transformation
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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