User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/06/28: Difference between revisions

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* All of the linkers are ~100 nM, which is what is expected for proper ligation.  
* 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 [http://openwetware.org/wiki/Corum:T4_Ligation ligation] with 2.5 μL backbone and 5 μL linkers.
* 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 [http://openwetware.org/wiki/Corum:T4_Ligation ligation] with 2.5 μL backbone and 5 μL linkers.
** I digested and then [http://openwetware.org/index.php?title=Corum:PCR_Purification 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.===
===Hypothesis 2: Gene L is necessary for phage propagation.===

Revision as of 08:37, 29 June 2012

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

  • Characterization C showed 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-PA-UTR1-NcoI linker = 23 nM
    • SphI-PB-UTR1-NcoI linker = 7 nM
    • SphI-PD-UTR1-NcoI linker = 18 nM
    • SphI-PF-UTR1-NcoI linker = 24 nM
    • SphI-PG-UTR1-NcoI linker = 10 nM
    • SphI-PL-UTR1-NcoI linker = 11 nM
    • SphI-PLPA-UTR1-NcoI linker = 4 nM
    • pBEST-SphI//NcoI-deGFP-T500 backbone = 10 nM
  • All of the linkers are ~10 nM, when they should be ~100 nM for proper ligation. Therefore, trashed these guys and repeated hybridization (for A, D, F) and PCR (for B, G, L, LA) to construct new linkers.
  • The backbone is at expected ~10 nM concentration, so I kept it for use in the next ligation.

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.

Training: Continuous system cell free expression.


Notes

Recently Edited Notebook Pages

Digital Signature

  • SC 19:09, 27 June 2012 (EDT):


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