IGEM:Groningen/Notebook/iGEM 2011/2011/09/06: Difference between revisions
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== | ==6-9-11== | ||
<br> | |||
<br> | |||
<br> Colony PCR of PcI-RBS-GFP-DT and K077002 | |||
<br> | |||
<br> Taq 10× buffer: 20μl | |||
<br> dNTPs 10mM: 4μl | |||
<br> MgCl2: 12μl | |||
<br> Forward Biobrick primer: 4μl | |||
<br> Reverse Biobrick primer: 4μl | |||
<br> Taq DNA polymerase: 1μl | |||
<br> MQ water: 155μl | |||
<br> | |||
<br> PCR conditions: | |||
<br> Preheated lid: 111°C | |||
<br> Denaturation: 94°C for 10 min. | |||
<br> Cycle 33×: | |||
<br> denaturation: 94°C for 30s. | |||
<br> annealing: 60°C for 30s. | |||
<br> Extension: 72°C for 2,5 min. | |||
<br> Final extension: 72°C for 10 min. | |||
<br> Store infinite at 4°C | |||
<br> | |||
<br> Analyse on a 1% TBE agarose gel. | |||
<br> | |||
<br> Cloning PBAD-RBS-GFP-DT, PBAD-cI-LVA, PBAD-cI-LVA-DT and PBAD-LasR-LVA-DT | |||
<br> Calculations were made with the Ligation calculator based on DNA concentrations according to the nanodrop | |||
<br> Digestion: | |||
<br> PBADaraC | |||
<br> 5μl insert (PBADaraC) | |||
<br> 1μl EcoRI | |||
<br> 1μl SpeI | |||
<br> 2μl FD buffer | |||
<br> 11μl MQ water | |||
<br> | |||
<br> RBS-GFP-DTvector: | |||
<br> 5μl vector | |||
<br> 1μl EcoRI | |||
<br> 1μl XbaI | |||
<br> 3μl FD buffer | |||
<br> 1μl FastAP | |||
<br> 19μl MQ | |||
<br> | |||
<br> cI-LVAvector: | |||
<br> 5μl vector | |||
<br> 1μl EcoRI | |||
<br> 1μl XbaI | |||
<br> 3μl FD buffer | |||
<br> 1μl FastAP | |||
<br> 19μl MQ | |||
<br> | |||
<br> PBAD-PSB1C3vector: | |||
<br> 5μl vector | |||
<br> 1μl SpeI | |||
<br> 1μl PstI | |||
<br> 3μl FD buffer | |||
<br> 1μl FastAP | |||
<br> 19μl MQ | |||
<br> | |||
<br> RBS-GFP-DT PCR product | |||
<br> 5μl insert (PBADaraC) | |||
<br> 1μl XbaI | |||
<br> 1μl PstI | |||
<br> 2μl FD buffer | |||
<br> 11μl MQ water | |||
<br> | |||
<br> cI-LVA PCR product | |||
<br> 3μl insert | |||
<br> 1μl XbaI | |||
<br> 1μl PstI | |||
<br> 2μl FD buffer | |||
<br> 13μl MQ water | |||
<br> | |||
<br> PBAD-cI-LVA vector: | |||
<br> 3μl vector | |||
<br> 1μl SpeI | |||
<br> 1μl PstI | |||
<br> 3μl FD buffer | |||
<br> 1μl FastAP | |||
<br> 21μl MQ | |||
<br> | |||
<br> PBAD-LasR-LVA vector: | |||
<br> 5μl vector | |||
<br> 1μl SpeI | |||
<br> 1μl PstI | |||
<br> 3μl FD buffer | |||
<br> 1μl FastAP | |||
<br> 19μl MQ | |||
<br> | |||
<br> DT product | |||
<br> 2μl insert | |||
<br> 1μl XbaI | |||
<br> 1μl PstI | |||
<br> 2μl FD buffer | |||
<br> 14μl MQ water | |||
<br> | |||
<br> Incubate the samples for 1h at 37 °C. | |||
<br> | |||
<br> DNA clean up with the High Pure PCR Purification Kit of Roche | |||
<br> | |||
<br> Ligation: | |||
<br> PBADaraC-RBS-GFP-DT | |||
<br> 5.8μl PBADaraC | |||
<br> 8.5μl vector RBS-GFP-DT | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 2.7μl MQ water | |||
<br> | |||
<br> PBADaraC-cI-LVA | |||
<br> 5.8μl PBADaraC | |||
<br> 8.5μl vector | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 2.7μl MQ water | |||
<br> | |||
<br> PBADaraC-RBS-GFP-DT | |||
<br> 5.4μl RBS-GFP-DT | |||
<br> 8.5μl vector PBAD-pSB1C3 | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 3.1μl MQ water | |||
<br> | |||
<br> PBADaraC-cI-LVA | |||
<br> 4μl cI-LVA | |||
<br> 8.5μl vector PBAD-pSB1C3 | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 4.5μl MQ water | |||
<br> | |||
<br> PBADaraC-cI-LVA-DT | |||
<br> 2.3μl DT | |||
<br> 8.5μl vector RBS-GFP-DT | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 6.2μl MQ water | |||
<br> | |||
<br> PBADaraC-LasR-LVA-DT | |||
<br> 2.3μl DT | |||
<br> 8.5μl vector RBS-GFP-DT | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 6.2μl MQ water | |||
<br> | |||
<br> Self ligation control: | |||
<br> 8.5μl vector | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 8.5μl MQ water | |||
<br> | |||
<br> Incubate the samples for 30 to 40 minutes at roomtemperature | |||
<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 | |||
<br> | |||
<br> Check PCR of autoinducing loop plasmids for Christoph and Vessa | |||
<br> | |||
<br> Taq 10× buffer: 52μl | |||
<br> dNTPs 10mM: 10.4μl | |||
<br> MgCl2: 31.2μl | |||
<br> Forward Biobrick primer: 10.4μl | |||
<br> Reverse Biobrick primer: 10.4μl | |||
<br> Taq DNA polymerase: 2.6μl | |||
<br> MQ water: 403μl | |||
<br> | |||
<br> PCR conditions: | |||
<br> Preheated lid: 111°C | |||
<br> Denaturation: 94°C for 10 min. | |||
<br> Cycle 30×: | |||
<br> denaturation: 94°C for 30s. | |||
<br> annealing: 60°C for 30s. | |||
<br> Extension: 72°C for 2,5 min. | |||
<br> Final extension: 72°C for 10 min. | |||
<br> Store infinite at 4°C | |||
<br> | |||
Revision as of 05:55, 6 September 2011
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6-9-11
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