IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 RNA Decoder/2010/06/30

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June 30, 2010

Today we tried cloning out MicA, MicF, OmpA, OmpF, and Hfq. The procedure we followed was:

  • 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 also ran controsl for each reaction: one without template, and one without polymerase. In each water replaced the missing material.

The PCR settings were:

  1. 95 C= 5 min.
  2. 95 C= 30 secs.
  3. 55 C= 30 secs.
  4. 72 C= 1 min.
  5. 72 C= 5 min.
  6. 4 C= Hold
  • Steps 2-4 repeated 30 cycles

We then ran a electrophoresis gel of my PCR results.