User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/07/25

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Hypothesis 2: Gene L is necessary for phage propagation.

  • From yesterday's whole plasmid PCR of ΦX174...
    • Quantifluore result showed 3-7 μg/μL DNA for the range of 0, 100, 200, 500, and 1000 nM. Assuming the DNA signal is coming from the 45-mer primer, I get the following results:
      • "100 nM" = 82 nM
      • "200 nM" = 215 nM
      • "500 nM" = 378 nM
      • "1000 nM" = 515 nM
    • CONCLUSION: No amplification and signal is from the 45-mer. Not sure why the plasmid didn't amplify. Will wait until PFU Ultra II DNAP and 33-mer primers come in before proceeding further.

Hypothesis 3: Gene L codes for a ~6 kDA protein.

  • After gel purification of band, performed ligation with pBEST-OR2OR1Pr-NcoI//XhoI-T500 and pBEST-OR2OR1Pr-NcoI//XhoI-T500 backbones and NcoI-COIL1-XhoI and NcoI-COIL2-XhoI linkers to create the following:
    • pBEST-OR2OR1Pr-UTR1-COIL1-T500
    • pBEST-OR2OR1Pr-UTR1-COIL2-T500
    • pBEST-Ptar-UTR1-COIL1-T500
    • pBEST-Ptar-UTR1-COIL2-T500