User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/07/10

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

• Ligation of the following SphI-PX-NheI linkers into pBEST-SphI//NheI-UTR1-deGFP-T500 ...
  1. SphI-PB-NheI
  2. SphI-PD-NheI
  3. SphI-PF-NheI
  4. SphI-PG-NheI
  5. SphI-PL-NheI
  6. SphI-PL-L-PA-NheI
  7. -linker control

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

• Ligation of the following SphI-PX-UTRX-NcoI linkers into pBEST-SphI//NcoI-deGFP-T500 ...
  1. SphI-PB-UTRB-NcoI
  2. SphI-PD-UTRD-NcoI
  3. SphI-PF-UTRF-NcoI
  4. SphI-PG-UTRG-NcoI
  5. SphI-PL-UTRL-NcoI
  6. -linker control

Hypothesis 2: Gene L is necessary for phage propagation.

• Problem with whole plasmid PCR is my primers.
• Primers amplified pWhitescript without any problem. Characterizing these using NEB Tm Calculator:
  • Sense primer: CCATGATTACGCCAAGCGCGCAATTAACCCTCAC, annealing T 64 °C, melting T 69 °C
  • Antisense primer: GTGAGGGTTAATTGCGCGCTTGGCGTAATCATGG, annealing T 64 °C, melting T 69 °C
• On the other hand, my primers for ΦX174 give:
  • ΦX174 sense primer: GATATTTTTCATGGTATTGATAAAGCTGTTGCCGATACTTGGAAC, annealing T 57 °C, melting T 63 °C
  • ΦX174 antisense primer: CTATAAAAAGTACCATAACTATTTCGACAACGGCTATGAACCTTG, annealing T 57 °C, melting T 62 °C
• WAIT! Antisense primer appears to be completely wrong, as it should the reverse compliment of the sense primer! Primers need to be reordered...
  • primer 1:
    1. GATATTTTTCATGGTATTGATAAAGCTGTTGCCGATACTTGGAAC, T_m = 63 °C, annealing T = 58 °C
    2. GTTCCAAGTATCGGCAACAGCTTTATCAATACCATGAAAAATATC, T_m = 63 °C
  • primer 2:
    1. GGTGTGGTTGATATTTTTCATGGTATTGATAAAGCTGTTGCCGATACTTGGAACAATTTCTGG, T_m = 69 °C, annealing T = 64 °C
    2. CCAGAAATTGTTCCAAGTATCGGCAACAGCTTTATCAATACCATGAAAAATATCAACCACACC, T_m = 69 °C
  • primer 3:
    1. GCTTCTGGTGTGGTTGATATTTTTCATGGTATTGATAAAGCTGTTGCCGATACTTGGAACAATTTCTGGAAAGACGG, T_m = °C, annealing T = °C
    2. CCGTCTTTCCAGAAATTGTTCCAAGTATCGGCAACAGCTTTATCAATACCATGAAAAATATCAACCACACCAGAAGC, T_m = °C

I ordered these primers from BMGC.