IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 RNA Decoder/2010/07/19

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July 19, 2010

We redid the PCR's from Saturday. The primers were not diluted to 1/10 their original concentrations.

  • 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
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