IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 RNA Decoder/2010/07/17

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July 17, 2010

Francis couldn't wait until Monday to get things rolling and came in on Saturday to get some work done. He did the following PCRs (and diluted the primers to 1/10 concentration):

  • RXN 1 (MicA)
    • 10 uL 10X Pfu Buffer
    • 0.4 uL Pfr DNA Polymerase
    • 0.4 uL Primer Forward
    • 0.4 uL Primer Reverse
    • 1.0 uL 4X dNTP
    • 10 uL MG1655 Template
    • 32.8 uL dH2O
    • Total= 50 uL
  • RXN 2 (OmpA)
    • 10 uL 10X Pfu Buffer
    • 0.4 uL Pfr DNA Polymerase
    • 0.4 uL Primer Forward
    • 0.4 uL Primer Reverse
    • 1.0 uL 4X dNTP
    • 10 uL MG1655 Template
    • 32.8 uL dH2O
    • Total= 50 uL
  • RXN 3 (MicF):
    • 10 uL 10X Pfu Buffer
    • 0.4 uL Pfr DNA Polymerase
    • 0.4 uL Primer Forward
    • 0.4 uL Primer Reverse
    • 1.0 uL 4X dNTP
    • 10 uL MG1655 Template
    • 32.8 uL dH2O
    • Total= 50 uL
  • RXN 4 (OmpF)
    • 10 uL 10X Pfu Buffer
    • 0.4 uL Pfr DNA Polymerase
    • 0.4 uL Primer Forward
    • 0.4 uL Primer Reverse
    • 1.0 uL 4X dNTP
    • 10 uL MG1655 Template
    • 32.8 uL dH2O
    • Total= 50 uL
  • RXN 5 (GadY)
    • 10 uL 10X Pfu Buffer
    • 0.4 uL Pfr DNA Polymerase
    • 0.4 uL Primer Forward
    • 0.4 uL Primer Reverse
    • 1.0 uL 4X dNTP
    • 10 uL MG1655 Template
    • 32.8 uL dH2O
    • Total= 50 uL
  • RXN 6 (GadX)
    • 10 uL 10X Pfu Buffer
    • 0.4 uL Pfr DNA Polymerase
    • 0.4 uL Primer Forward
    • 0.4 uL Primer Reverse
    • 1.0 uL 4X dNTP
    • 10 uL MG1655 Template
    • 32.8 uL dH2O
    • Total= 50 uL
  • RXN 7(Hfq)
    • 10 uL 10X Pfu Buffer
    • 0.4 uL Pfr DNA Polymerase
    • 0.4 uL Primer Forward
    • 0.4 uL Primer Reverse
    • 1.0 uL 4X dNTP
    • 10 uL MG1655 Template
    • 32.8 uL dH2O
    • Total= 50 uL
  • RXN 8 (EGFP)
    • 10 uL 10X Pfu Buffer
    • 0.4 uL Pfr DNA Polymerase
    • 0.4 uL Primer Forward
    • 0.4 uL Primer Reverse
    • 1.0 uL 4X dNTP
    • 1 uL PXG-10 Template
    • 41.8 uL dH2O
    • Total= 50 uL
  • RXN 9 (Polymerase Control)
    • 10 uL 10X Pfu Buffer
    • 0.4 uL Primer Forward
    • 0.4 uL Primer Reverse
    • 1.0 uL 4X dNTP
    • 10 uL MG1655 Template
    • 33.2 uL dH2O
    • Total= 50 uL
  • RXN 10 (Template Control)
    • 10 uL 10X Pfu Buffer
    • 0.4 uL Pfr DNA Polymerase
    • 0.4 uL Primer Forward
    • 0.4 uL Primer Reverse
    • 1.0 uL 4X dNTP
    • 42.8 uL dH2O
    • Total= 50 uL
  • RXN 11 (Primer Control)
    • 10 uL 10X Pfu Buffer
    • 0.4 uL Pfr DNA Polymerase
    • 1.0 uL 4X dNTP
    • 10 uL MG1655 Template
    • 33.6 uL dH2O
    • Total= 50 uL


The PCR setting that was used:

  1. 95 C: 5 min.
  2. 95 C: 30 sec.
  3. 51 C: 30 sec.
  4. 72 C: 1.5 min.
  5. 72 C: 10 min.
  6. 4 C: hold

Numbers 2-4 were repeated 32 times.


The Concentrations of the templates were:

  • PXG-10: 475 ng/uL
  • MG1655: 2.8 ng/uL

The Concentrations of the cloned genes were:

  • Hfq: 44.70 ng/uL
  • OmpA: 1.66 ng/uL
  • GadY: 15.88 ng/uL
  • OmpF: 2.65 ng/uL

EGFP: -1.47 ng/uL MicF: 16.78 ng/uL MicA: 7.60 ng/uL GadX: 43.69 ng/uL