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Cell Free Expression of PHIX174: June 21, 2012
- SC 16:49, 21 June 2012 (EDT):
Characterization B: Expression of PHIX174 promoters fused to UTR1-deGFP.
- Five colonies were observed from yesterday's transformation of pBEST-PA-UTR1-deGFP-T500 ligation products into JM109.
- Prepared 2.5 mL standard minicultures from these colonies for overnight incubation.
Characterization C: Expression of PHIX174 promoters fused to UTRX-deGFP.
- Yesterday, I performed PCR to create precursors to SphI-(B, G, L, and "PL-L-PA")-NcoI linkers. Today, I performed digestion with SphI and NcoI without performing PCR purification first. Perhaps I will be able to eliminate an unnecessary step. Just to be sure, I only used half of each 50 μL PCR sample.
- Reaction conditions: NEB Buffer 4 1X + BSA 1mg/ml in 33.75 μL reaction volume at 37 °C overnight.
- In parallel, to create vector backbone with complementary sticky ends, I digested 67nM pBEST-OR2OR1Pr-UTR1-deGFP-T500. Reaction conditions: 6 μL template, 1 μL NEB Buffer 4 10X, 1 μL BSA 10mg/ml, 1 μL SphI, 1 μL NcoI in 10 μL reaction volume at 37 °C overnight.
Hypothesis 2: Gene L is necessary for phage propagation.
- Need to design new primers (regular and mutagenic) for amplifying PHIX174 genome.
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