IGEM:Groningen/Notebook/iGEM 2011/2011/07/12

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12-07-11
Few colonies were seen on the plates of yesterday's transformation. Checking transformants of yesterday with colony PCR: Mastermix (9+1 samples): 10× Taq buffer: 20μl dNTP mix 10mM: 4μl MgCl2: 12μl taq 5u/μl: 1μl Forward primer BB vector 10μM: 4μl Reverse primer BB vector 10μM: 4μl MilliQ water: 155μl PCR conditions: 1. Pre heated lid: 111 °C 2. Denaturation: 10 min. at 94°C 3. Cycle 33×: Denaturation: 30s at 94°C Annealing:   30s at 60°C Extension:   2 min. at 72°C 4. Final extension: 10 min. at 72°C 5. Store infinite at 4°C Another transformation was done with the ligation mixtures of yesterday (were stored overnight in the fridge at 4°C) cI-LVA, LasR-LVA and pSB1A3-DT were digested, ligated and transformed again like yesterday, but the digestion and ligation time were increased to 1 hour. Colony PCR results: two colonies of cI-LVA seems to contain the right construct (a band of around 1000bp was seen on the 1% agarose gel and this should be the size of the product with the BB vector primers). Colonies are grown overnight in LB medium with ampicillin (1μg/ml). Also the pSB1A3 vector with RBS-GFP-DT are grown overnight, since there is no more plasmid available in our storage.


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