User:Andrew Perry/Notebook/bbGibson/2011/06/08

bbGibson assembly

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bbGibson PCR tests This is the first attempt at using the new bbGibson primers to amplify some standard Biobrick vectors and parts. Used my standard PCR protocol with Phusion polymerase. 98 deg, 30 sec 3 cycles 98 deg, 20 sec 55 deg, 30 sec 72 deg, 4 min 28 cycles 98 deg, 20 sec 65 deg, 30 sec 72 deg, 4 min 72 deg, 5 min 12 deg, (overnight) Agarose gel of PCR reactions amplifying standard Biobrick vectors and some parts with new bbGibson primers. Stained with EZ-vision (Amresco). Lane 1. Mass Ruler (Fermentas), 2. pSB1T3 (linear PCR template) V.f/V.r, 3. pSB1C3 (circular plasmid template) V.f/V.r, 4. I13522 (tetR-RBS-GFP-tt) A.f/ END.r, 5. I13522 (tetR-RBS-GFP-tt) A.f/ A.r, 6. I13521 (tetR-RBS-RFP-tt) B.f/ END.r. Result: Got product of expected size for every part except I13522 with B.f/END.r primers (lane 6). Bad miniprep template DNA ? Gel purified products (Sigma GenElute kit).