User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/08/23

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Hypothesis 2: Gene L is necessary for phage propagation.

  • Here is the quantiluore results from the WP-PCR of 0.1 nM ΦX174 with variable elongation time. Time is is min:sec:
    1. 10min/3^4 = 00:07 - 7.9 nM (3%)
    2. 10min/3^3 = 00:22 - 10.9 nM (2%)
    3. 10min/3^2 = 01:07 - 16.2 nM (2%)
    4. 10min/3^1 = 03:20 - 40.1 nM (1.2%)
    5. 10min/3^0 = 10:00 - 21.3 nM (0.4%)
    6. "0.1" nM ΦX174 - 0.093 nM (37%)
  • Everything looks good, except the 10:00 sample is only about half the 3:20 sample, whereas I would expect it to be no less than the 3:20 sample. I will repeat the elongation time experiment, except with the following changed variables values for Ta: 0 min, 1, 2, 3, 4, 5, and 10.
  • 70 μL WP-PCR:
    • 42.3 μL H2O
    • 8.75 μL 2mM dNTPs (each) (0.25mM each final)
    • 7.7 μL 10X reaction buffer
    • 7.7 μL 10 μM primer 4 mix
    • 2.19 μL 3.2 nM ΦX174 template (0.1 nM final)
    • 1.40 μL PfuUltra II fusion HS DNA polymerase
  • Aliquot 7×10μL.
  • Cycling parameters:
    1. 95 °C 2 min
    2. 95 °C 20 s
    3. 58 °C 20 s
    4. 72 °C X min, where X = 0, 1, 2, 3, 4, 5, 10
    5. Repeat 2-4 an additional 29 times = 30 cycles
    6. 12 °C hold

  • Next, I will re-optimize primer concentration over the range 1mM/10^x, where x = 1, 2, 3, 4, 5, and ∞ (in reverse order, this is equivalent to 0, 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM). 60 μL + 10% WP-PCR reaction:
    • 34.3 μL H2O
    • 6.6 μL 10X reaction buffer
    • 8.25 μL 2mM dNTPs (each) (0.25mM each final)
    • 2.063 μL 3.2 nM ΦX174 template (0.1 nM final)
    • 1.32 μL PfuUltra II fusion HS DNA polymerase
  • Aliquot 6×9 μL and add 10X primer range (0, 100 nM, 1 μM, 10 μM, 100 μM, and 1 mM).
  • Cycling parameters:
    1. 95 °C 2 min
    2. 95 °C 20 s
    3. 58 °C 20 s
    4. 72 °C ??? s
    5. Repeat 2-4 an additional 29 times = 30 cycles
    6. 72 °C 3 min
    7. 12 °C hold
  • Things that still need to be optimized:
    1. Ta = 54, 56, 58, 60, 62 °C
    2. N cycles = 0, 10, 20, 25, 30, 35, 40, 50
  • After that, tasks include characterizing effects of:
    1. parallel PFU ligation
    2. DpnI digestion (w/ and w/o PCR purification)
    3. Topisomerase IV / Gyrase (preceded by PCR purification) - how to assay linking number?
  • Final experiment is planned to be WP-PCR of 0.1 nM ΦX174 at optimized conditions (primer concentration, Ta, elongation time, N), + PFU ligase
    • experimental 1 = +template, +primer 4, +DNAP
    • experimental 2 = +template. +primer 4 T3585A, +DNAP
    • control 1: -template, +primer 4, +DNAP
    • control 2: -template, +primer 4 T3485, +DNAP
    • control 3: +template, +primer 4, +DNAP
    • control 4: +template, +primer 4 T3485, +DNAP
    • control 5: +template, -primers, +DNAP
  • Followed by:
    • (possible purification and then) DpnI digestion
    • PCR purification
    • Linking number adjustment by gyrase / topisomerase IV
    • PCR purification

Hypothesis 2: Gene L is necessary for phage propagation.

  • Here are the quantiluore results from the WP-PCR of 0.1 nM ΦX174 with variable primer concentration.
    1. 0 nM - nM ( %)
    2. 10 nM - nM ( %)
    3. 100 nM = 00:22 - nM ( %)
    4. 1 μM = 01:07 - nM ( %)
    5. 10 μM = 03:20 - nM ( %)
    6. 100 μM = 10:00 - nM ( %)
  • Optimizing N, the number of PCR cycles. 50 μL WP-PCR reaction:
    • 62.4 μL H2O
    • 6.25 μL 2mM dNTPs (each) (0.25mM each final)
    • 5 μL 10X reaction buffer
    • 5 μL ??? μM primer 4 mix
    • 1.563 μL 3.2 nM ΦX174 template (0.1 nM final)
    • 1 μL PfuUltra II fusion HS DNA polymerase
  • Cycling parameters:
    1. 95 °C 2 min
    2. 95 °C 20 s
    3. 58 °C 20 s
    4. 72 °C ??? s
    5. Repeat 2-4 an additional 39 times = 40 cycles
    6. 72 °C 3 min
    7. 12 °C hold
  • 5 μL removed every N cycles for N = 0, 5, 10, 15, 20, 25, 30, 35, 40 (9 samples)
  • Things that still need to be optimized:
    1. Ta = 54, 56, 58, 60, 62 °C
  • After that, tasks include characterizing effects of:
    1. parallel PFU ligation
    2. DpnI digestion (w/ and w/o PCR purification)
    3. Topisomerase IV / Gyrase (preceded by PCR purification) - how to assay linking number?
  • Final experiment is planned to be WP-PCR of 0.1 nM ΦX174 at optimized conditions (primer concentration, Ta, elongation time, N), + PFU ligase
    • experimental 1 = +template, +primer 4, +DNAP
    • experimental 2 = +template. +primer 4 T3585A, +DNAP
    • control 1: -template, +primer 4, +DNAP
    • control 2: -template, +primer 4 T3485, +DNAP
    • control 3: +template, +primer 4, +DNAP
    • control 4: +template, +primer 4 T3485, +DNAP
    • control 5: +template, -primers, +DNAP
  • Followed by:
    • (possible purification and then) DpnI digestion
    • PCR purification
    • Linking number adjustment by gyrase / topisomerase IV
    • PCR purification
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