User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/06/29: Difference between revisions

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** SphI-PG-UTR1-NcoI linker
** SphI-PG-UTR1-NcoI linker
** SphI-PL-UTR1-NcoI linker
** SphI-PL-UTR1-NcoI linker
** SphI-PLpa-UTR1-NcoI linker
** SphI-PLPA-UTR1-NcoI linker
* All of the linkers should be at ~100 nM final concentration, so they can be used with the pBEST-SphI//NcoI-deGFP-T500 linker (10 nM) in ligation to create the desired set of pBEST-PX-UTR1-deGFP-T500 constructs.
* All of the linkers should be at ~100 nM final concentration, so they can be used with the pBEST-SphI//NcoI-deGFP-T500 linker (10 nM) in ligation to create the desired set of pBEST-PX-UTR1-deGFP-T500 constructs.
===Characterization C: Expression of PHIX174 promoters fused to UTRX-deGFP.===
===Characterization C: Expression of PHIX174 promoters fused to UTRX-deGFP.===


Revision as of 08:51, 29 June 2012

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

  • I peformed hybridization on oligonucleotides to construct the following:
    • SphI-PA-UTR1-NcoI linker at 100 nM final concentration
    • SphI-PD-UTR1-NcoI linker at 100 nM final concentration
    • SphI-PF-UTR1-NcoI linker at 100 nM final concentration
  • I peformed PCR followed by PCR purification, speedvac down to ~20 μL, digestion with SphI and NcoI, and another round of PCR purification to create the following linkers:
    • SphI-PB-UTR1-NcoI linker
    • SphI-PG-UTR1-NcoI linker
    • SphI-PL-UTR1-NcoI linker
    • SphI-PLPA-UTR1-NcoI linker
  • All of the linkers should be at ~100 nM final concentration, so they can be used with the pBEST-SphI//NcoI-deGFP-T500 linker (10 nM) in ligation to create the desired set of pBEST-PX-UTR1-deGFP-T500 constructs.

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

  • There were no colonies after transformation. Therefore, I decided to characterize all linkers and backbones for those constructs as well as the UTR1-deGFP constructs via quantifluore DNA quantification. Results were as follows:
    • SphI-null-NheI linker = 73 nM
    • SphI-PA-UTRA-NheI linker = 46 nM
    • SphI-PB-UTRB-NheI linker = 37 nM
    • SphI-PD-UTRD-NheI linker = 66 nM
    • SphI-PF-UTRF-NheI linker = 66 nM
    • SphI-PG-UTRG-NheI linker = 139 nM
    • SphI-PL-UTRL-NheI linker = 87 nM
    • SphI-PLPA-UTRA-NheI raw PCR product = 166 nM
    • pBEST-SphI//NheI -deGFP-T500 backbone = 2 nM
  • All of the linkers are ~100 nM, which is what is expected for proper ligation.
  • The backbone is at expected 2 nM concentration but is needed at ~10 nM concentration, so to obtain a proper ligation with 1:100 backbone to linker concentration, the volume of backbone needs to be increased by a factor of 5 in ligation. I performed ligation with 2.5 μL backbone and 5 μL linkers.
    • I digested and then PCR purified the SphI-PLPA-UTRA-NheI raw PCR product with SphI and NheI overnight to create the SphI-PLPA-UTRA-NheI linker (yet again)

Hypothesis 2: Gene L is necessary for phage propagation.

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


Notes

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  • SC 11:50, 29 June 2012 (EDT):
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