User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/06/27: Difference between revisions

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==Entry title==
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===Characterization B: Expression of PHIX174 promoters fused to UTR1-deGFP.===
* Today, I digested yesterday's PCR product and pBEST-OR2OR1Pr-UTR1-deGFP-T500 with SphI and NheI for 3 hr.
* I then gel extracted these two products to give the SphI-PL-PA-NheI linker and the pBEST-SphI//NheI-UTR1-deGFP-T500 backbone, respectively.
** Gel electrophoresis bands were as expected: <500b observed for SphI-PL-PA-NheI (312b expected) and ~> 3000b observed for pBEST-SphI//NheI-UTR1-deGFP-T500 (3096 expected), using marker 17.
===Characterization C: Expression of PHIX174 promoters fused to UTRX-deGFP.===
* No colonies were observed from yesterday's transformation. My next step will be to verify the linker and backbone with gel electrophoresis and quantifluore.
===Hypothesis 2: Gene L is necessary for phage propagation.===
* Need to design new primers (regular and mutagenic) for amplifying PHIX174 genome.
===Training: Continuous system cell free expression.===


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==Notes==
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==Digital Signature==
*'''SC 19:09, 27 June 2012 (EDT)''':


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Revision as of 16:09, 27 June 2012

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

  • Today, I digested yesterday's PCR product and pBEST-OR2OR1Pr-UTR1-deGFP-T500 with SphI and NheI for 3 hr.
  • I then gel extracted these two products to give the SphI-PL-PA-NheI linker and the pBEST-SphI//NheI-UTR1-deGFP-T500 backbone, respectively.
    • Gel electrophoresis bands were as expected: <500b observed for SphI-PL-PA-NheI (312b expected) and ~> 3000b observed for pBEST-SphI//NheI-UTR1-deGFP-T500 (3096 expected), using marker 17.

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

  • No colonies were observed from yesterday's transformation. My next step will be to verify the linker and backbone with gel electrophoresis and quantifluore.

Hypothesis 2: Gene L is necessary for phage propagation.

  • Need to design new primers (regular and mutagenic) for amplifying PHIX174 genome.

Training: Continuous system cell free expression.


Notes

Recently Edited Notebook Pages

Digital Signature

  • SC 19:09, 27 June 2012 (EDT):


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