User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/06/20

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Cell Free Expression of PHIX174: June 20, 2012

SC 21:03, 19 June 2012 (EDT):

Characterization B: Expression of PHIX174 promoters fused to UTR1-deGFP.

  • Following yesterday's ligation to (hopefully) form pBAD-PA-UTR1-deGFP-T500, I transformed 3 μL ligation product into JM109 competent cells and incubated overnight.

Characterization C: Expression of PHIX174 promoters fused to UTRX-deGFP.

  • The oligonucleotides for creating PX-UTRX linkers were received today. The file specifying their construction is here.
  • For B, G, L, and "PL-L-PA", I performed standard PCR using PHIX174 genomic DNA as template, TH = 58 °C, and 30 s elongation time. Linkers will be created tomorrow upon digestion and gel extraction of the PCR products with SphI and NcoI.
  • On that note, tomorrow I'll have to digest pBEST-... in parallel to obtain pBEST-SphI//NcoI-deGFP-T500 vector backbone.

Hypothesis 2: Gene L is necessary for phage propagation.

  • I ran yesterday's QuickchangeII whole plasmid PCR products on a gel. Results were as follows:
    • DNAP- Control: no band observed (nothing expected)
    • pWhitescript 4.5kb control plasmid: X observed (4.5kb ccDNA expected)


  • None.

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