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

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

• I peformed hybridization on oligonucleotides to construct the following:
  • SphI-PA-NcoI linker at 100 nM final concentration
  • SphI-PD-NcoI linker at 100 nM final concentration
  • SphI-PF-NcoI linker at 100 nM final concentration
• I peformed PCR followed by PCR purification, speedvac down to ~20 μL, digestion with SphI and NcoI, and another round of PCR purification to create the following linkers:
  • SphI-PB-NcoI linker
  • SphI-PG-NcoI linker
  • SphI-PL-NcoI linker
  • SphI-PLPA-NcoI linker
• All of the linkers should be at ~100 nM final concentration, so they can be used with the pBEST-SphI//NcoI-deGFP-T500 linker (10 nM) in ligation to create the desired set of pBEST-PX-UTR1-deGFP-T500 constructs.

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 linker ~ 100 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.

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 create 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-UTR1-NcoI linker
    2. SphI-PG-UTR1-NcoI linker
    3. SphI-PL-UTR1-NcoI linker
    4. SphI-PLPA-UTRA-NheI
  • Ligate 0.5 μL pBEST-SphI//NcoI-UTR1-deGFP-T500 (10nM) with 5 μL linkers (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 2.5 μL pBEST-SphI//NheI-deGFP-T500 (2nM) with 5 μL linkers SphI-PX-UTRX-NheI (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
  • Miniprep the minicultures. Submit samples for sequencing ((4 × 15 = 60 total).

Notes

Recent changes

Digital Signature

• SC 11:50, 29 June 2012 (EDT):