Characterization B: Expression of PHIX174 promoters fused to UTR1-deGFP.
- While I was away, Fillipo attempted to construct these. Either transformants didn't grow, or the negative control ligation showed colonies.
- Started again by making pBEST-SphI//NcoI-deGFP-T500 linker by digesting 67nM stock pBEST-OR2OR1Pr-UTR1-deGFP-T500 with SphI and NcoI (20 μL total volume), following by gel extraction.
Characterization C: Expression of PHIX174 promoters fused to UTRX-deGFP.
- While I was away, Fillipo attempted to construct these. Either transformants didn't grow, or the negative control ligation showed colonies.
- Started again by making pBEST-SphI//NheI-deGFP-T500 linker by digesting 67nM stock pBEST-OR2OR1Pr-UTR1-deGFP-T500 with SphI and NcoI (30 μL total volume), following by gel extraction.
Hypothesis 2: Gene L is necessary for phage propagation.
- Previously, I demonstrated that pWhitescript could be amplified by whole plasmid PCR using the control primers included in the Quickchange II kit. Template- NC showed no amplification. Following the manual's protocol exactly, I made a 50 μL PFU PCR mix that included everything except primers and and then divided it by three. In the three tubes, I placed template (0.1 nmol final) and primers (200nM final):
- Negative control: ΦX174 sense and antisense primers
- Reaction: ΦX174 template, ΦX174 sense and antisense primers
- Positive control: pWhitescript template, pWhitescript sense and antisense primers
- Thermal cycling was exactly as specified in the manual, with a 10 min elongation step and 20 total cycles of PCR.
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PHIX174 Cell Free Expression
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