User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/07/05: Difference between revisions

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===Hypothesis 2: Gene L is necessary for phage propagation.===
===Hypothesis 2: Gene L is necessary for phage propagation.===


* Need to design new primers (regular and mutagenic) for amplifying PHIX174 genome.
* Previously, I demonstrated that pWhitescript could be amplified by whole plasmid PCR using the control primers included in the [http://openwetware.org/wiki/Image:Quickchange_II.pdf 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 [http://openwetware.org/wiki/Image:Quickchange_II.pdf manual], with a 10 min elongation step and 20 total cycles of PCR.




Revision as of 13:55, 5 July 2012

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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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