Haynes Lab:Notebook/Engineering PC-TFs/2012/11/06

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Summary

  • Gibson Assembly
  • Check with PCR

Assembly 1 contains PCR rxns 1,2, and 3. Assembly 2 contains PCR rxns 4,5. There is no addition of vector this time.

1 2
Gibson Rxn mix 15 μL 15 μL
DNA fragments 5 μL 5 μL



DNA fragments 1 2
#1 1.0 μL
#2 1.0 μL
#3 1.0 μL
#4 1.0 μL
#5 1.0 μL
dH2O 2.0 μL 3.0 μL
Total 5.0 μL 5.0 μL






PCR Check

  1. Product 1: outer-most primers for whole assembly
  2. Product 1: Gibson primer pair for hPCD
  3. Product 2: outer-most primers
  4. Product 2: Gibson primer pair for hPCD


Reagent 1 2 3 4
Gibson product 0.5 μL 0.5 0.5 0.5
primer 1 1.0 1.0 1.0 1.0
primer 2 1.0 1.0 1.0 1.0
2x GoTaq PCR mix 14.5 14.5 14.5 14.5
dH2O 12.0 12.0 12.0 12.0



Program:

  • 95°C, 3 min.
  • [95°C, 30 sec; 57°C, 30 sec; 72°C, 30 sec] 30 cycles
  • 72°C, 3 min.
  • 4°C ∞


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