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

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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

Recent changes

20 October 2017

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BME100 f2017:Group5 W0800 L4‎‎ (5 changes | history) . . (+62). . [Anna Hoge‎ (5×)]

     

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(cur | prev) . . (+1). . Anna Hoge (talk | contribs) (SNP Information & Primer Design)

     

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     09:23  Carrico‎ (diff | hist) . . (-305). . Yar (talk | contribs) (use mediawiki markup for images, not raw html)
     09:15 (User creation log) . . User account Msupestka (talk | contribs) was created by Yar (talk | contribs) and password was sent by email ‎

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Beauchamp:DataSharing‎‎ (2 changes | history) . . (+228). . [John Magnotti‎ (2×)]

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     07:49  Min-Ho Kim Lab:Lab Members‎ (diff | hist) . . (-89). . Min-Ho Kim (talk | contribs)

19 October 2017

     21:07  BME100 f2017:Group7 W0800 L4‎ (diff | hist) . . (+977). . Miguel R. Almanza Lopez (talk | contribs) (SNP Information & Primer Design)

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BME100 f2017:Group6 W1030 L4‎‎ (4 changes | history) . . (+1,447). . [Ray Gerard Regorgo‎ (4×)]

     

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(cur | prev) . . (-196). . Ray Gerard Regorgo (talk | contribs) (OUR TEAM)

     

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Rich Lab:Funding‎‎ (2 changes | history) . . (-12). . [Isabel C. Morales‎ (2×)]

     

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     14:58  CONJ606:Materials‎ (diff | hist) . . (+685). . Peteral (talk | contribs) (Week 2: Me Myself and I-dentifiers, The Key To Immortality (Persistence))

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User:Peteral‎‎ (2 changes | history) . . (+536). . [Peteral‎ (2×)]

     

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(cur | prev) . . (+185). . Peteral (talk | contribs) (Alec's Discussion Questions)

     

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(cur | prev) . . (+351). . Peteral (talk | contribs) (Alec's Discussion Questions)

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BME100 f2017:Group14 W0800 L4‎‎ (9 changes | history) . . (+9). . [Shae M. Diaz‎ (9×)]

     

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(cur | prev) . . (+8). . Shae M. Diaz (talk | contribs) (Protocol)

     

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(cur | prev) . . (-31). . Shae M. Diaz (talk | contribs) (TEAM MEMBERS)

     

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(cur | prev) . . (+31). . Shae M. Diaz (talk | contribs) (TEAM MEMBERS)

     

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(cur | prev) . . (+1). . Shae M. Diaz (talk | contribs) (TEAM MEMBERS)

     

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 m   14:12  Swartz‎ (diff | hist) . . (+61). . Marcus Rohovie (talk | contribs)
     14:10 (Move log) . . Marcus Rohovie (talk | contribs) moved page Swartz to Swartz:Main

     14:08 

(Upload log). .

[Thorntca‎; Shae M. Diaz‎ (3×)]

     

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. . Shae M. Diaz (talk | contribs) uploaded File:Shaediaz100.JPG

     

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Klenow Assembly Method: Seamless cloning‎‎ (3 changes | history) . . (+101). . [David M.D. Bailey‎ (3×)]

     

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(cur | prev) . . (-5). . David M.D. Bailey (talk | contribs) (The Klenow Assembly Method (KAM) for the seamless cloning of overlapping double stranded DNA fragments: Very cheap alternative to the Gibson Assembly)

     

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(cur | prev) . . (+1). . David M.D. Bailey (talk | contribs) (The Klenow Assembly Method (KAM) for the seamless cloning of overlapping double stranded DNA fragments: Very cheap alternative to the Gibson Assembly)

     

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(cur | prev) . . (+105). . David M.D. Bailey (talk | contribs) (The Klenow Assembly Method (KAM) for the seamless cloning of overlapping double stranded DNA fragments: Very cheap alternative to the Gibson Assembly)

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User:Thorntca‎‎ (8 changes | history) . . (+529). . [Thorntca‎ (8×)]

     

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Digital Signature

  • SC 11:50, 29 June 2012 (EDT):