User:Eric M. Walters/Notebook/Spring 2012/2012/05/07

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Cyanobacterial acrylate production Main project page
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Increased acrylate toxicity with MCR

  • Still waiting on colonies to come up

In the mean time, will start from the other direction: acsA KO, then put MCR in

acsA::accBCDA in wt 7002

  • Transforming wt 7002 (OD = 1.62) with the acsA::accBCDA construct I made 4/16.
    • Reserving some (11 μL) to amplify more construct off of.
    • Usually want OD 1-1.5, but will err on the side of it being high.
  • I'm adding 23 μL at 135 ng/μL (3,105 ng)
  • Adding it to 2 mL wt 7002, also putting 2 mL of wt 7002 in a tube alongside it as negative control.

Making more of the acsA::accBCDA

4 x 50 ul reactions

  • 10 μL 5x HF Phusion
  • 1 μL acsA-UpF-v2
  • 1 μL acsA-DnR-v2
  • 1 μL dNTPs
  • 1 μL template DNA (full construct)
  • 1 μL Phusion
  • 35 μL H2O

1:30 extension, 72°C annealing


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