IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 RNA Decoder/2010/08/04

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August 4, 2010

I did mini-preps of the 5 inoculations from yesterday to check for the insert in the plasmids of the transformed cells. I also took concentrations for each of the 5 samples and they were:

  • Reaction A: 93.0 ng/uL
  • Reaction B: 68.7 ng/uL
  • Reaction C: 116.4 ng/uL
  • Reaction D: 128.6 ng/uL
  • Reaction E: 54.4 ng/uL

I also checked for succesful PCR amplification of the inserts with VR and VF2:

  • Reaction A
    • 1.0 uL template
    • 5.0 uL 10X pfu buffer
    • 0.4 uL Primer F
    • 0.4 uL Primer R
    • 0.4 uL Turbo Polymerase
    • 1.0 uL dNTP
    • 41.8 uL dH2O
  • Reaction B
    • 1.0 uL template
    • 5.0 uL 10X pfu buffer
    • 0.4 uL Primer F
    • 0.4 uL Primer R
    • 0.4 uL Turbo Polymerase
    • 1.0 uL dNTP
    • 41.8 uL dH2O
  • Reaction C
    • 1.0 uL template
    • 5.0 uL 10X pfu buffer
    • 0.4 uL Primer F
    • 0.4 uL Primer R
    • 0.4 uL Turbo Polymerase
    • 1.0 uL dNTP
    • 41.8 uL dH2O
  • Reaction D
    • 1.0 uL template
    • 5.0 uL 10X pfu buffer
    • 0.4 uL Primer F
    • 0.4 uL Primer R
    • 0.4 uL Turbo Polymerase
    • 1.0 uL dNTP
    • 41.8 uL dH2O
  • Reaction E
    • 1.0 uL template
    • 5.0 uL 10X pfu buffer
    • 0.4 uL Primer F
    • 0.4 uL Primer R
    • 0.4 uL Turbo Polymerase
    • 1.0 uL dNTP
    • 41.8 uL dH2O
  • Template Control
    • 5.0 uL 10X pfu buffer
    • 0.4 uL Primer F
    • 0.4 uL Primer R
    • 0.4 uL Turbo Polymerase
    • 1.0 uL dNTP
    • 42.8 uL dH2O

The PCR setting that was used: 95 C: 3 min. 95 C: 30 sec. 51 C: 30 sec. 72 C: 45 sec. 72 C: 5 min. 4 C: hold Numbers 2-4 were repeated 32 times.



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