User:Karmella Haynes/Notebook/BioBrick cloning/2013/04/12

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04/12/13

  • Chem. Competent cell prep: CaCl wash; 15% glycerol/CaCl resuspension (used 30 mL instead of 20 mL); incubate in cold room on ice o/n
  • MV9 building: colony PCR



MV9 building: colony PCR

  • Pick 8 colonies from plates 1, 2, 3, and 4; make a mini-streak library, then wash pipette tip in PCR master mix
  • Pick 4 colonies from plate 5 (neg. ctrl)
  • Primers for MV2 vector: DD122B (fwd), DD123B (rev); see BBa_J176122
Reagent Volume x38 Expected:
MV2 (empty) = 177
MV9 (single insert) = 253
extra inserts = +76
DNA (colony) --- ---
10 μM primer 1 1.0 38.0
10 μM primer 2 1.0 38.0
2x GoTaq green 10.0 380.0
dH2O 8.0 304.0
  20 μL  

Thermal cycling

  • 95°C, 3 min.
  • [95°C, 15 sec; 57°C, 15 sec; 72°C, 15 sec] x30
  • 72°C, 3 min.
  • 4°C, ∞



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