User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/05

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01/05/13

  • Golden gate: PCR parts, complete protocol for Brady's construct
  • Gibson: PCR parts, complete protocol for Brady's construct



Golden Gate Assembly

  • Try new protocol from Dave Savage
  1. hPCD + BL01 + V0120
  2. hPCD + BL01 + pSB1A3
  • Check the sequences for BsmBI sites

PCR

  1. V0120, gg_? / gg_?
  2. pSB1A3, gg_? / gg_?
  3. hPCD, gg_BBP-hPCD F / gg_hPCD R
  4. BL01, gg_BL01 F / gg_BL01-BBS R
Reagent Volume Expected:
1. V0120 = size
2. pSB1A3 = size
3. hPCD = size
4. BL01 = size
DNA(plasmid) 0.5 μL
10 μM primer 1 1.0
10 μM primer 2 1.0
2x GoTaq mix 25.0
dH2O ##
  50 μL
  • 95°C, 3 min.
  • [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 2 min.] x 30
  • 72°C, 3 min.
  • 4°C, ∞