IGEM:Groningen/Notebook/iGEM 2011/2011/06/24: Difference between revisions
Joyce Mulder (talk | contribs) (Autocreate 2011/06/24 Entry for IGEM:Groningen/Notebook/iGEM_2011) |
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== | ==24-6-11== | ||
<br> | |||
<br> Purify the PCR product with the High Pure PCR Purification kit from Roche | |||
<br> PhybB-RBS-GFP-DT | |||
<br> | |||
<br> Digestion mix: | |||
<br> 18μl buffer | |||
<br> 4.5μl EcoRI | |||
<br> 4.5μl SpeI | |||
<br> 49μl MQ water | |||
<br> | |||
<br> Vector mix: | |||
<br> 18μl buffer | |||
<br> 4.5μl EcoRI | |||
<br> 4.5μl XbaI | |||
<br> 49μl MQ water | |||
<br> | |||
<br> Ligation: | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 7μl insert | |||
<br> 3μl vector | |||
<br> 7μl MQ water | |||
<br> | |||
<br> Ligate 2 hours at room temperature | |||
<br> | |||
<br> Transformation | |||
<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 | |||
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Revision as of 03:39, 21 September 2011
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24-6-11
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