User:Eric M. Walters/Notebook/Spring 2012/2012/02/07

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Round 2: Constructing acsA_up-accBCDA-acsA_down

Amplifying the three parts

  • 100 uL reactions of each (50x2)
  • Done a la 1/27

acsA_up & acsA_down

  • 1 ul forward primer (300 uM stock)
  • 1 ul reverse primer (300 uM stock)
  • 1 ul PCC 7002 gDNA
  • 1 ul dNTP mix
  • 1 ul Phusion
  • 10 ul HF buffer
  • 35 ul Water


  1. 98°C for 2:00
  2. 98°C for :30
  3. 60°C for :30
  4. 72°C for :15
    1. Cycle 35x
  5. 72°C for 4:00
  6. 4°C forever

accBCDA

  • 1 ul forward primer (300 uM stock)
  • 1 ul reverse primer (300 uM stock)
  • 1 ul pΨ223 (100 uM stock)
  • 1 ul dNTP mix
  • 1 ul Phusion
  • 10 ul HF buffer
  • 35 ul Water


  1. 98°C for 2:00
  2. 98°C for :30
  3. 60°C for :30
  4. 72°C for 1:10
    1. Cycle 35x
  5. 72°C for 4:00
  6. 4°C forever