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

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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.
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).


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