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

From OpenWetWare
Jump to navigationJump to search
Line 19: Line 19:
* 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. Negative control sub water for linker.
** [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. Negative control sub water for linker.


Revision as of 17:29, 5 July 2012

PHIX174 Cell Free Expression <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

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. 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. 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.



Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Sean_P_Corum/Notebook/PHIX174_Cell_Free/2012/07/05&oldid=612675"