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

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


* I peformed hybridization on oligonucleotides to construct the following:
* Reboot and starting construction over from scratch.
** SphI-PA-NheI linker at 100 nM final concentration
** SphI-PD-NheI linker at 100 nM final concentration
** SphI-PF-NheI linker at 100 nM final concentration
* I peformed [http://openwetware.org/index.php?title=Corum:PCR PCR] followed by [http://openwetware.org/index.php?title=Corum:PCR_Purification PCR purification], speedvac down to ~20 μL, digestion with SphI and NcoI, and another round of [http://openwetware.org/index.php?title=Corum:PCR_Purification PCR purification] to create the following linkers:
** SphI-PB-NheI linker
** SphI-PG-NheI linker
** SphI-PL-NheI linker
** SphI-PLPA-NheI linker
* All of the linkers should be at ~100 nM final concentration, so they can be used with the pBEST-SphI//NheI-UTR1-deGFP-T500 linker (2nM) 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 12:42, 29 June 2012

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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.

  • 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-NcoI linker = 73 nM
    • SphI-PA-UTRA-NcoI linker = 46 nM
    • SphI-PB-UTRB-NcoI linker = 37 nM
    • SphI-PD-UTRD-NcoI linker = 66 nM
    • SphI-PF-UTRF-NcoI linker = 66 nM
    • SphI-PG-UTRG-NcoI linker = 139 nM
    • SphI-PL-UTRL-NcoI linker = 87 nM
    • SphI-PLPA-UTRA-NcoI linker ~ 100 nM
    • pBEST-SphI//NcoI -deGFP-T500 backbone = 10 nM
  • All of the linkers are ~100 nM, which is what is expected for proper ligation.
  • Performed ligation with 0.5 μL backbone and 5 μL linkers.

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-NheI linker
      2. SphI-PD-NheI linker
      3. SphI-PF-NheI linker
      • Hydridize 2mM complimentary ssDNA oligonucleotides. Product is linker at 1mM concentration. Perform 1/10 serial dilutions to 100 nM final concentration.
    • To create the following linkers:
      1. SphI-PB-NheI linker
      2. SphI-PG-NheI linker
      3. SphI-PL-NheI linker
      4. 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 NheI.
  • Today +1
    • To obtain following linkers, perform Gel extraction (elute in 100 μL H2O, linkers should be at ~100nM concentration):
      1. SphI-PB-UTR1-NheI linker
      2. SphI-PG-UTR1-NheI linker
      3. SphI-PL-UTR1-NheI linker
      4. SphI-PLPA-UTRA-NheI
    • Ligate 2.5 μL pBEST-SphI//NheI-UTR1-deGFP-T500 (2nM) with 5 μL linkers SphI-PX-NheI (including hybridization products from yesterday) to create the following constructs:
      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
    • Ligate 0.5 μL pBEST-SphI//NcoI-deGFP-T500 (10nM) with 5 μL linkers SphI-PX-UTRX-NcoI (X = null, A, B, D, F, G, L, 'PLPA'; ~100nM) to create the following constructs:
      1. pBEST-null-deGFP-T500
      2. pBEST-PA-UTRA-deGFP-T500
      3. pBEST-PB-UTRB-deGFP-T500
      4. pBEST-PD-UTRD-deGFP-T500
      5. pBEST-PF-UTRF-deGFP-T500
      6. pBEST-PG-UTRG-deGFP-T500
      7. pBEST-PL-UTRL-deGFP-T500
    • 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 +2
    • Transform the ligation products into JM109 competent cells and grow overnight (15 plates).
  • Today +3
    • Select 4 colonies per plate and grow 2.5 mL miniculures (4 × 15 = 60 total).
  • Today +4&5


Notes

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Digital Signature

  • SC 11:50, 29 June 2012 (EDT):
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