IGEM:Groningen/Notebook/iGEM 2011/2011/08/23

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23-8-11 Change of cloning strategy: Instead of cloning 3 times, I can do a procedure with only 2 cloning steps since I already have the constructs PhybB and PBAD in a chloramphenicol vector. So what I am going to do is: Clone the RBS-cI-LVA and RBS-LasR-LVA in the vector with already PhybB and PBAD sequences, but than you have to clone downstream those promotors the protein sequences. That is why you digest the vector with SpeI and PstI and the PCR products with XbaI and PstI. If the transformation plates look well and the colony PCR can be done and show that there are some clones having the right fragmentsize, thursday the plasmids can be isolated and the second cloning procedure can be done: cloning downstream the double terminator sequence. So if it all goes well, the problem will be fixed on friday PCR on templates with cI-LVA and LasR-LVA: Composition per reaction: 10× pfu buffer with MgSO4: 5μl dNTPs 10mM: 1μl Forward primer: 1μl Reverse : 1μl Template: 1μl Pfu polymerase: 1μl MQ water: 40μl PCR conditions: Pre heated lid: 111°C Hotstart Denaturation: 94°C for 5min Cycle (33×) Denaturation: 94°C for 30s Annealing: 69°C for 30s Extenstion: 72°C for 2min Final extension: 72°C for 10min Store infinite at 4°C Analyse PCR samples on a 1% agarosegel Digestion cI-LVA 10μl insert 1μl XbaI 1μl PstI 2μl FD buffer 6μl MQ water LasR-LVA 14μl insert 1μl XbaI 1μl PstI 2μl FD buffer 2μl MQ water PBAD-pSB1C3 vector 4μl vector 1μl SpeI 1μl PstI 3μl FD buffer 1μl FastAP 20μl MQ water PhybB-pSB1C3 vector 4μl vector 1μl SpeI 1μl PstI 3μl FD buffer 1μl FastAP 20μl MQ water Incubate for 60 min at 37 degrees DNA clean up: Ligation: PBAD-RBS-cI-LVA-pSB1C3 8.5μl vector 5.6μl insert 2μl T4 DNA ligase buffer 1μl T4 DNA ligase 2.9μl MQ water PBAD-RBS-LasR-LVA-pSB1C3 8.5μl vector 6.7μl insert 2μl T4 DNA ligase buffer 1μl T4 DNA ligase 1.8μl MQ water PhybB-RBS-cI-LVA-pSB1C3 8.5μl vector 7μl insert 2μl T4 DNA ligase buffer 1μl T4 DNA ligase 1.5 μl MQ water PhybB-RBS-LasR-LVA-pSB1C3 8.5μl vector 8.5μl insert 2μl T4 DNA ligase buffer 1μl T4 DNA ligase Incubate for 30-40 minutes 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 PCR for the double terminator part: Composition per reaction: 10× pfu buffer with MgSO4: 5μl dNTPs 10mM: 1μl Forward primer: 1μl Reverse : 1μl Template(pSB1A3DT): 1μl Pfu polymerase: 1μl MQ water: 40μl 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 1.5min Final extension: 72°C for 15min Store infinite at 4°C Analyse PCR samples on a 1% agarosegel