User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/06

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01/06/13

  • Gibson Assembly: pick colonies & check via colony PCR
  • Media: Make LB agar +amp
  • Gibson Assembly: redo transformation with more DNA


Gibson Assembly results

  • hPCD + BL01 + V0120, 2μL 1x Gibson: no colonies for assembly (1) or no-insert control (2)
  • hPCD + BL01 + pSB1A3, 2μL 1x Gibson: 1 colony for assembly (3), none on control (4)
  • hPCD + BL01 + V0120, 2μL 1/4x Gibson: 2 colonies for assembly (5), 1 on control (6)
  • hPCD + BL01 + pSB1A3, 2μL 1/4x Gibson: 1 colony for assembly (7), 2 on control (8)
  • Pick and streak colonies from plates 3, 5, 7
  • Use same pipette tip for colony PCR
Reagent Volume 5x mix PCR gel
30 μL/lane, 1% agarose; Ladder
Colony --- ---
10 μM primer BL9 1.0 5.0
10 μM primer BL10 1.0 5.0
2x GoTaq 12.5 62.5
dH2O 10.5 52.5
  25.0 μL

PCR

  • 95°C, 3 min.
  • [95°C, 30 sec.; 95°C, 30 sec.; 72°C, 1 min.] x35
  • 72°C, 3 min.
  • 4°C, ∞

Conclusion: looks like primer dimer. Re-do the transformation with 3x more DNA (from yesterday, stored at -20°C)


Transformation

  1. hPCD + BL01 + V0120, 6.0 μL 1/4x Gibson
  2. V0120, 6.0 μL 1/4x Gibson
  3. hPCD + BL01 + pSB1A3, 6.0 μL 1/4x Gibson
  4. pSB1A3, 6.0 μL 1/4x Gibson
  • Add DNA to 50 μL DH5α-Turbo; ice/ 10 min.
  • Plate on 100 μg/mL amp.



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