User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/06/27

PHIX174 Cell Free Expression

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Characterization B: Expression of PHIX174 promoters fused to UTR1-deGFP. Today, I digested yesterday's PCR product and pBEST-OR2OR1Pr-UTR1-deGFP-T500 with SphI and NheI for 3 hr. I then gel extracted these two products to give the SphI-PL-PA-NheI linker and the pBEST-SphI//NheI-UTR1-deGFP-T500 backbone, respectively. Gel electrophoresis bands as: nothing observed for SphI-PL-PA-NheI (312b expected) and ~ 3000b observed for pBEST-SphI//NheI-UTR1-deGFP-T500 (3096 expected), using marker 17. PCR for SphI-PL-PA-NheI linker repeated. Characterization C: Expression of PHIX174 promoters fused to UTRX-deGFP. No colonies were observed from yesterday's transformation. My next step will be to verify the linker and backbone with gel electrophoresis and quantifluore. Hypothesis 2: Gene L is necessary for phage propagation. Need to design new primers (regular and mutagenic) for amplifying PHIX174 genome. Training: Continuous system cell free expression.

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• SC 19:09, 27 June 2012 (EDT):