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
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PHIX174 Cell Free Expression
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