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

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PHIX174 Cell Free Expression Main project page
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Characterization B: Expression of PHIX174 promoters fused to UTR1-deGFP.

  • Reboot and starting construction over from scratch.

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

  • Reboot and starting construction over from scratch.

Hypothesis 2: Gene L is necessary for phage propagation.

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

To do

  • Today
    • To create the following linkers:
      1. SphI-PA-NcoI linker
      2. SphI-PD-NcoI linker
      3. SphI-PF-NcoI linker
      • Hydridize 2mM complimentary ssDNA oligonucleotides. Product is linker at 1mM concentration. Perform 1/10 serial dilutions to 100 nM final concentration.
    • To initiate the following linkers:
      1. SphI-PB-NcoI linker
      2. SphI-PG-NcoI linker
      3. SphI-PL-NcoI linker
      4. SphI-PLPA-NcoI linker
      • Perform PCR using ΦX174 genomic DNA as template.
      • Follow with PCR purification. Elute with 100 μL H2O and speedvac to ~20 μL.
      • Double digest overnight with SphI and NcoI.
  • Today +1
    • To obtain following linkers, perform Gel extraction (elute in 100 μL H2O, linkers should be at ~100nM concentration):
      1. SphI-PB-NcoI linker
      2. SphI-PG-NcoI linker
      3. SphI-PL-NcoI linker
      4. SphI-PLPA-NcoI linker
    • To create the following linkers:
      1. SphI-PA-NheI linker
      2. SphI-PB-NheI linker
      3. SphI-PD-NheI linker
      4. SphI-PF-NheI linker
      5. SphI-PG-NheI linker
      6. SphI-PL-NheI linker
      • Hydridize 2mM complimentary ssDNA oligonucleotides. Product is linker at 1mM concentration. Perform 1/10 serial dilutions to 100 nM final concentration.
    • To initiate the following linkers:
      1. SphI-PLPA-NheI linker
      • Perform PCR using ΦX174 genomic DNA as template.
      • Follow with PCR purification. Elute with 100 μL H2O and speedvac to ~20 μL.
      • Double digest overnight with SphI and NcoI.
  • Today +3
    • To obtain following linkers, perform Gel extraction (elute in 100 μL H2O, linkers should be at ~100nM concentration):
      1. SphI-PLPA-NheI linker
    • The entire list of linkers is now as follows:
      1. SphI-PA-NcoI
      2. SphI-PB-NcoI
      3. SphI-PD-NcoI
      4. SphI-PF-NcoI
      5. SphI-PG-NcoI
      6. SphI-PL-NcoI
      7. SphI-PLPA-NcoI
      8. SphI-PA-UTRA-NheI
      9. SphI-PB-UTRB-NheI
      10. SphI-PD-UTRD-NheI
      11. SphI-PF-UTRF-NheI
      12. SphI-PG-UTRG-NheI
      13. SphI-PL-UTRL-NheI
      14. SphI-PLPA-UTRA-NheI
      15. SphI-NULL-NheI
    • Verify that linker concentrations are all ~100nM with quantifluore DNA quantification.
    • The list of backbones is:
      1. pBEST-SphI//NcoI-UTR1-deGFP-T500 (10nM). Ligate 0.5 μL with 5 μL SphI-PX-NcoI linkers.
      2. pBEST-SphI//NheI-deGFP-T500 (2nM) Ligate 0.5 μL with 5 μL SphI-PX-UTRX-NheI linkers.
    • The entire list of ligation product constructs is now:
      1. pBEST-PA-UTR1-deGFP-T500
      2. pBEST-PB-UTR1-deGFP-T500
      3. pBEST-PD-UTR1-deGFP-T500
      4. pBEST-PF-UTR1-deGFP-T500
      5. pBEST-PG-UTR1-deGFP-T500
      6. pBEST-PL-UTR1-deGFP-T500
      7. pBEST-PLPA-UTR1-deGFP-T500
      8. pBEST-PA-UTRA-deGFP-T500
      9. pBEST-PB-UTRB-deGFP-T500
      10. pBEST-PD-UTRD-deGFP-T500
      11. pBEST-PF-UTRF-deGFP-T500
      12. pBEST-PG-UTRG-deGFP-T500
      13. pBEST-PL-UTRL-deGFP-T500
      14. pBEST-PLPA-UTRA-deGFP-T500
      15. pBEST-NULL-deGFP-T500
  • Today +4
    • Transform the ligation products into JM109 competent cells and grow overnight (15 plates).
  • Today +5
    • Select 4 colonies per plate and grow 2.5 mL miniculures (4 × 15 = 60 total).
  • Today +6&7


Notes

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Digital Signature

  • SC 11:50, 29 June 2012 (EDT):
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