User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/06/21: Difference between revisions

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


* [http://openwetware.org/wiki/User:Sean_P_Corum/Notebook/PHIX174_Cell_Free/2012/06/20 Yesterday's] [http://openwetware.org/wiki/Corum:DNA_Hybridization DNA hybridization] of A, D, and F linkers wasn't going well because of leaky tubes. Instead, I tried an alternative protocol, DNA hybridization in a thermocycler.
* [xhttp://openwetware.org/wiki/User:Sean_P_Corum/Notebook/PHIX174_Cell_Free/2012/06/20 Yesterday], I performed PCR to create precursors to SphI-(B, G, L, and "PL-L-PA")-NcoI linkers. Today, I performed digestion with SphI and NcoI without performing PCR purification first. Perhaps I will be able to eliminate an unnecessary step. Just to be sure, I only used half of each 50 μL PCR sample.
* [Yesterday http://openwetware.org/wiki/User:Sean_P_Corum/Notebook/PHIX174_Cell_Free/2012/06/20], I performed PCR to create precursors to SphI-(B, G, L, and "PL-L-PA")-NcoI linkers. Today, I performed digestion with SphI and NcoI without performing PCR purification first. Perhaps I will be able to eliminate an unnecessary step. Just to be sure, I only used half of each 50 μL PCR sample.
** Reaction conditions: NEB Buffer 4 1X + BSA 1mg/ml in 33.75 μL reaction volume at 37 °C overnight.
** Reaction conditions: NEB Buffer 4 1X + BSA 1mg/ml in 33.75 μL reaction volume at 37 °C overnight.
** In parallel, to create vector backbone with complementary sticky ends, I digested 67nM pBEST-OR2OR1Pr-UTR1-deGFP-T500. Reaction conditions: 6 μL template, 1 μL NEB Buffer 4 10X, 1 μL BSA 10mg/ml, 1 μL SphI, 1 μL NcoI in 10 μL reaction volume at 37 °C overnight.
** In parallel, to create vector backbone with complementary sticky ends, I digested 67nM pBEST-OR2OR1Pr-UTR1-deGFP-T500. Reaction conditions: 6 μL template, 1 μL NEB Buffer 4 10X, 1 μL BSA 10mg/ml, 1 μL SphI, 1 μL NcoI in 10 μL reaction volume at 37 °C overnight.


===Hypothesis 2: Gene L is necessary for phage propagation.===
===Hypothesis 2: Gene L is necessary for phage propagation.===

Revision as of 13:03, 25 June 2012

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Cell Free Expression of PHIX174: June 21, 2012

  • SC 16:49, 21 June 2012 (EDT):

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

  • Five colonies were observed from yesterday's transformation of pBEST-PA-UTR1-deGFP-T500 ligation products into JM109.
  • Prepared 2.5 mL standard minicultures from these colonies for overnight incubation.

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

  • [xhttp://openwetware.org/wiki/User:Sean_P_Corum/Notebook/PHIX174_Cell_Free/2012/06/20 Yesterday], I performed PCR to create precursors to SphI-(B, G, L, and "PL-L-PA")-NcoI linkers. Today, I performed digestion with SphI and NcoI without performing PCR purification first. Perhaps I will be able to eliminate an unnecessary step. Just to be sure, I only used half of each 50 μL PCR sample.
    • Reaction conditions: NEB Buffer 4 1X + BSA 1mg/ml in 33.75 μL reaction volume at 37 °C overnight.
    • In parallel, to create vector backbone with complementary sticky ends, I digested 67nM pBEST-OR2OR1Pr-UTR1-deGFP-T500. Reaction conditions: 6 μL template, 1 μL NEB Buffer 4 10X, 1 μL BSA 10mg/ml, 1 μL SphI, 1 μL NcoI in 10 μL reaction volume at 37 °C overnight.

Hypothesis 2: Gene L is necessary for phage propagation.

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


Notes

  • None.

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