IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 RNA Decoder/2010/07/01

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July 1, 2010

We ran another PCR for MicA, MicF, OmpA, OmpF, and Hfq. We also ran two controls for the experiment: one without polymerase and one without template. Their uL amounts were replaced with dH2O. The procedure we followed:

  • 10X Buffer 5.0 uL
  • Pfu Turbo DNA Polymerase 0.4 uL
  • Primer Forward 0.5 uL
  • Primer Reverse 0.5 uL
  • dNTP 1.0 uL
  • Template (MG1655) 0.5 uL
  • dH2O 42.1 uL
  • Total= 50 uL

A master mix instead of adding each of those amounts to each tube. The master mix was made as such (MM for 5 RXNs):

  • 10X Buffer 25uL
  • Pfu Turbo DNA Polymerase 2 uL
  • dNTP 5 uL
  • Template (MG1655) 2.5 uL
  • dH2O 210.5 uL

We then ran the PCR at these settings:

  1. 95 C: 5 mins.
  2. 95 C: 30 sec.
  3. 58 C: 30 sec.
  4. 72 C: 1 min.
  5. 4 C: hold
  • Steps 2-4 were repeated 32 cycles.

Once the PCR was complete we ran a gel for the trials.