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

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m (→Characterization C: Expression of PHIX174 promoters fused to UTRX-deGFP.)
Current revision (20:32, 5 July 2012) (view source)
m (→Characterization C: Expression of PHIX174 promoters fused to UTRX-deGFP.)
 
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* Started again by making pBEST-SphI//NcoI-deGFP-T500 linker by [http://openwetware.org/wiki/Corum:Digestion digesting] 67nM stock pBEST-OR2OR1Pr-UTR1-deGFP-T500 with SphI and NcoI (20 μL total volume), following by [http://openwetware.org/wiki/Corum:Gel_Purification gel extraction].
* Started again by making pBEST-SphI//NcoI-deGFP-T500 linker by [http://openwetware.org/wiki/Corum:Digestion digesting] 67nM stock pBEST-OR2OR1Pr-UTR1-deGFP-T500 with SphI and NcoI (20 μL total volume), following by [http://openwetware.org/wiki/Corum:Gel_Purification gel extraction].
** Gel showed a line at 2.5 kb as expected. Uncut plasmid DNA sample showed farther migration and higher order species, as expected.
** Gel showed a line at 2.5 kb as expected. Uncut plasmid DNA sample showed farther migration and higher order species, as expected.
 +
** [pBEST-SphI//NcoI-deGFP-T500] = 15 nM, via [http://openwetware.org/wiki/Corum:DNA_Quantification quantifluore DNA quantification].
 +
** [http://openwetware.org/wiki/Corum:T4_Ligation Ligated] 0.5 μL pBEST-SphI//NcoI-deGFP-T500 backbone and 5 μL 100nM SphI-PA-UTR1-NcoI linker (ratio ~1:67). Negative control sub water for linker.
===Characterization C: Expression of PHIX174 promoters fused to UTRX-deGFP.===
===Characterization C: Expression of PHIX174 promoters fused to UTRX-deGFP.===
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* Started again by making pBEST-SphI//NheI-deGFP-T500 linker by [http://openwetware.org/wiki/Corum:Digestion digesting] 67nM stock pBEST-OR2OR1Pr-UTR1-deGFP-T500 with SphI and NheI (30 μL total volume), following by [http://openwetware.org/wiki/Corum:Gel_Purification gel extraction].
* Started again by making pBEST-SphI//NheI-deGFP-T500 linker by [http://openwetware.org/wiki/Corum:Digestion digesting] 67nM stock pBEST-OR2OR1Pr-UTR1-deGFP-T500 with SphI and NheI (30 μL total volume), following by [http://openwetware.org/wiki/Corum:Gel_Purification gel extraction].
** Gel showed a line at 2.5 kb as expected. Uncut plasmid DNA sample showed farther migration and higher order species, as expected.
** Gel showed a line at 2.5 kb as expected. Uncut plasmid DNA sample showed farther migration and higher order species, as expected.
-
** [pBEST-SphI//NheI-deGFP-T500] = nM, via [http://openwetware.org/wiki/Corum:DNA_Quantification quantifluore DNA quantification].
+
** [pBEST-SphI//NheI-deGFP-T500] = 16 nM, via [http://openwetware.org/wiki/Corum:DNA_Quantification quantifluore DNA quantification].
 +
** [http://openwetware.org/wiki/Corum:T4_Ligation Ligated] 0.5 μL pBEST-SphI//NheI-deGFP-T500 backbone and 5 μL 100nM SphI-PA-UTRA-NheI linker (ratio ~1:62). Negative control sub water for linker.
===Hypothesis 2: Gene L is necessary for phage propagation.===
===Hypothesis 2: Gene L is necessary for phage propagation.===
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** Reaction: ΦX174 template, ΦX174 sense and antisense primers
** Reaction: ΦX174 template, ΦX174 sense and antisense primers
** Positive control: pWhitescript template, pWhitescript 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.
+
** 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.

Current revision

PHIX174 Cell Free Expression Main project page
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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.
    • Gel showed a line at 2.5 kb as expected. Uncut plasmid DNA sample showed farther migration and higher order species, as expected.
    • [pBEST-SphI//NcoI-deGFP-T500] = 15 nM, via quantifluore DNA quantification.
    • Ligated 0.5 μL pBEST-SphI//NcoI-deGFP-T500 backbone and 5 μL 100nM SphI-PA-UTR1-NcoI linker (ratio ~1:67). Negative control sub water for linker.

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 NheI (30 μL total volume), following by gel extraction.
    • Gel showed a line at 2.5 kb as expected. Uncut plasmid DNA sample showed farther migration and higher order species, as expected.
    • [pBEST-SphI//NheI-deGFP-T500] = 16 nM, via quantifluore DNA quantification.
    • Ligated 0.5 μL pBEST-SphI//NheI-deGFP-T500 backbone and 5 μL 100nM SphI-PA-UTRA-NheI linker (ratio ~1:62). Negative control sub water for linker.

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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