IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 RNA Decoder/2010/07/20

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July 20, 2010

Today we retried the EGFP PCR because we were not able to clone it out yet. We did two of the same reactions for the EGFP:

  • RXN 1 EGFP (Done twice in two separate tubes)
    • 0.5 uL Template (pXG-10 475 ng/uL)
    • 5.0 uL 10X pfu Buffer
    • 0.4 uL Primer 52820847 (1/10 dilution)
    • 0.4 uL Primer 52820848 (1/10 dilution)
    • 1.0 uL 4X dNTP
    • 0.2 uL pfu DNA polymerase (Turbo)
    • 42.5 uL dH2O
    • Total: 50 uL


  • RXN 3 Control 1 (no template)
    • 5.0 uL 10X pfu Buffer
    • 0.4 uL Primer 52820847 (1/10 dilution)
    • 0.4 uL Primer 52820848 (1/10 dilution)
    • 1.0 uL 4X dNTP
    • 0.2 uL pfu DNA polymerase (Turbo)
    • 43.0o uL dH2O
    • Total: 50 uL


  • RXN 4 Control 2 (no primers)
    • 0.5 uL Template (pXG-10 475 ng/uL)
    • 5.0 uL 10X pfu Buffer)
    • 1.0 uL 4X dNTP
    • 0.2 uL pfu DNA polymerase (Turbo)
    • 43.3 uL dH2O
    • Total: 50 uL


  • RXN 5 Control 3 (No polymerase)
    • 0.5 uL Template (pXG-10 475 ng/uL)
    • 5.0 uL 10X pfu Buffer
    • 0.4 uL Primer 52820847 (1/10 dilution)
    • 0.4 uL Primer 52820848 (1/10 dilution)
    • 1.0 uL 4X dNTP
    • 42.7 uL dH2O
    • Total: 50 uL


The PCR machines settings were:

  1. 95 C: 5 mins.
  2. 95 C: 30 secs.
  3. 55 C: 30 secs.
  4. 72 C: 1 min.
  5. 72 C: 10 mins.
  6. 4 C: hold

Numbers 2-4 were repeated 34 times.