IGEM:Groningen/Notebook/iGEM 2011/2011/07/12: Difference between revisions
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== | ==12-07-11== | ||
<br> Very few colonies were seen on the plates of yesterday's transformation. | |||
<br> Checking transformants of yesterday with colony PCR: | |||
<br>Mastermix (9+1 samples): | |||
<br> 10× Taq buffer: 20μl | |||
<br> dNTP mix 10mM: 4μl | |||
<br> MgCl2: 12μl | |||
<br> taq 5u/μl: 1μl | |||
<br> Forward primer BB vector 10μM: 4μl | |||
<br> Reverse primer BB vector 10μM: 4μl | |||
<br> MilliQ water: 155μl | |||
<br> PCR conditions: | |||
<br> 1. Pre heated lid: 111 °C | |||
<br> 2. Denaturation: 10 min. at 94°C | |||
<br> 3. Cycle 33×: | |||
<br> Denaturation: 30s at 94°C | |||
<br> Annealing: 30s at 60°C | |||
<br> Extension: 2 min. at 72°C | |||
<br> 4. Final extension: 10 min. at 72°C | |||
<br> 5. Store infinite at 4°C | |||
<br> | |||
<br> Another transformation was done with the ligation mixtures of yesterday (were stored overnight in the fridge at 4°C) | |||
<br> 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. | |||
<br> Colony PCR results: two colonies of cI-LVA seems to contain the right construct (a band of around 1000bp was seen on the 1% <br>agarose gel and this should be the size of the product with the BB vector primers). | |||
<br> Colonies are grown overnight in LB medium with ampicillin (1μg/ml). Also the pSB1A3 vector with RBS-GFP-DT are grown <br>overnight, since there is no more plasmid available in our storage. | |||
Revision as of 07:12, 12 July 2011
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