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

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* Primers amplified pWhitescript without any problem. Characterizing these using [http://www.neb.com/nebecomm/tech_reference/TmCalc/Default.asp#.T_3_w3ixm18 NEB Tm Calculator]:
* Primers amplified pWhitescript without any problem. Characterizing these using [http://www.neb.com/nebecomm/tech_reference/TmCalc/Default.asp#.T_3_w3ixm18 NEB Tm Calculator]:
** Sense primer: CCATGATTACGCCAAGCGCGCAATTAACCCTCAC, annealing T 64 °C, melting T 69 °C
** Sense primer: CCATGATTACGCCAAGCGCGCAATTAACCCTCAC, annealing T 64 °C, melting T 69 °C
** Antisense 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:
* On the other hand, my primers for ΦX174 give:
** Sense primer: GATATTTTTCATGGTATTGATAAAGCTGTTGCCGATACTTGGAAC, annealing T 57 °C, melting T 63 °C
** ΦX174 sense primer 1: GATATTTTTCATGGTATTGATAAAGCTGTTGCCGATACTTGGAAC, annealing T 57 °C, melting T 63 °C
** Antisense primer: CTATAAAAAGTACCATAACTATTTCGACAACGGCTATGAACCTTG, annealing T 57 °C, melting T 62 °C
** ΦX174 antisense primer: CTATAAAAAGTACCATAACTATTTCGACAACGGCTATGAACCTTG, annealing T 57 °C, melting T 62 °C
* Supposing that temperature is the problem, it appears I need to increase annealing and melting temperatures of my primers to 64 °C and 69 °C, respectively.  
* Supposing that temperature is the problem, it appears I need to increase annealing and melting temperatures of my primers to 64 °C and 69 °C, respectively.
** ΦX174 sense primer 1: CTGGTGTGGTTGATATTTTTCATGGTATTGATAAAGCTGTTGCCGATACTTGGAACAATTTCTGG, annealing T 57 °C, melting T 63 °C
** ΦX174 antisense primer: CTATAAAAAGTACCATAACTATTTCGACAACGGCTATGAACCTTG, annealing T 57 °C, melting T 62 °C
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__NOTOC__
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Revision as of 16:16, 11 July 2012

PHIX174 Cell Free Expression <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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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 1: GATATTTTTCATGGTATTGATAAAGCTGTTGCCGATACTTGGAAC, annealing T 57 °C, melting T 63 °C
    • ΦX174 antisense primer: CTATAAAAAGTACCATAACTATTTCGACAACGGCTATGAACCTTG, annealing T 57 °C, melting T 62 °C
  • Supposing that temperature is the problem, it appears I need to increase annealing and melting temperatures of my primers to 64 °C and 69 °C, respectively.
    • ΦX174 sense primer 1: CTGGTGTGGTTGATATTTTTCATGGTATTGATAAAGCTGTTGCCGATACTTGGAACAATTTCTGG, annealing T 57 °C, melting T 63 °C
    • ΦX174 antisense primer: CTATAAAAAGTACCATAACTATTTCGACAACGGCTATGAACCTTG, annealing T 57 °C, melting T 62 °C
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