User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/08/24: Difference between revisions

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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 and 1 μMM primer 4 (each) with variable elongation time. Time is is min:sec:
*# 0:00 - 4.8 nM (1.3%)
*# 1:00 - 8.6 nM (0.4%)
*# 2:00 - 20.4 nM (1.0%)
*# 3:00 - 30.6 nM (0.04%)
*# 4:00 - 27.8 nM (2%)
*# 5:00 - 44.3 nM (4%)
*# 10:00 - 47.9 nM (2%)
* Obviously, the advice to elongate for 15 s / kb from the Pfu Ultra II DNAP handbook is no good. If I followed that advice, I'd elongate the ΦX174 genome (5386 bp) for 90 s and achieve ~14.g nM yield, whereas when I elongated for 10 min, I achieveded 47.9 nM, a 3.3-fold increase.  Therefore, I will fix the elongation parameter to 2 min / kb and adjust the WP-PCR protocol to elongate for 12 min for ΦX174. I also not that the 4:00 sample seems to be mis-measured, since the rest of the data "looks like it makes sense" when plotted together.
* Next, testing variable elongation temperature: T<sup>e</sup> = 66, 68, 70, 72, 74 °C
* 50 μL WP-PCR:
** 30.2 μL H<sub>2</sub>O
** 6.25 μL 2mM dNTPs (each) (0.25mM each final)
** 5.5 μL 10X reaction buffer
** 5.5 μL 10 μM primer 4 mix
** 1.563 μL 3.2 nM ΦX174 template (0.1 nM final)
** 1 μL PfuUltra II fusion HS DNA polymerase
* Aliquot 5×10μL.
* Cycling parameters:
*# 95 °C 2 min
*# 95 °C 20 s
*# 58 °C 20 s
*# X °C 10 min, where X = 66, 68, 70, 72, 74
*# Repeat 2-4 an additional 29 times = 30 cycles
*# 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 H<sub>2</sub>O
** 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:
*# 95 °C 2 min
*# 95 °C 20 s
*# 58 °C 20 s
*# 72 °C ??? s
*# Repeat 2-4 an additional 29 times = 30 cycles
*# 72 °C 3 min
*# 12 °C hold
* Things that still need to be optimized:
*# T<sub>a</sub> = 54, 56, 58, 60, 62 °C
*# N cycles = 0, 10, 20, 25, 30, 35, 40, 50
* After that, tasks include characterizing effects of:
*# parallel PFU ligation
*# DpnI digestion (w/ and w/o PCR purification)
*# 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, T<sub>a</sub>, 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
* Here are the quantiluore results from the WP-PCR of 0.1 nM ΦX174 with variable cycle number, N.
* Here are the quantiluore results from the WP-PCR of 0.1 nM ΦX174 with variable cycle number, N.
*# 0 -  nM ( %)
*# 0 -  nM ( %)

Revision as of 09:21, 25 August 2012

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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 and 1 μMM primer 4 (each) with variable elongation time. Time is is min:sec:
    1. 0:00 - 4.8 nM (1.3%)
    2. 1:00 - 8.6 nM (0.4%)
    3. 2:00 - 20.4 nM (1.0%)
    4. 3:00 - 30.6 nM (0.04%)
    5. 4:00 - 27.8 nM (2%)
    6. 5:00 - 44.3 nM (4%)
    7. 10:00 - 47.9 nM (2%)
  • Obviously, the advice to elongate for 15 s / kb from the Pfu Ultra II DNAP handbook is no good. If I followed that advice, I'd elongate the ΦX174 genome (5386 bp) for 90 s and achieve ~14.g nM yield, whereas when I elongated for 10 min, I achieveded 47.9 nM, a 3.3-fold increase. Therefore, I will fix the elongation parameter to 2 min / kb and adjust the WP-PCR protocol to elongate for 12 min for ΦX174. I also not that the 4:00 sample seems to be mis-measured, since the rest of the data "looks like it makes sense" when plotted together.
  • Next, testing variable elongation temperature: Te = 66, 68, 70, 72, 74 °C
  • 50 μL WP-PCR:
    • 30.2 μL H2O
    • 6.25 μL 2mM dNTPs (each) (0.25mM each final)
    • 5.5 μL 10X reaction buffer
    • 5.5 μL 10 μM primer 4 mix
    • 1.563 μL 3.2 nM ΦX174 template (0.1 nM final)
    • 1 μL PfuUltra II fusion HS DNA polymerase
  • Aliquot 5×10μL.
  • Cycling parameters:
    1. 95 °C 2 min
    2. 95 °C 20 s
    3. 58 °C 20 s
    4. X °C 10 min, where X = 66, 68, 70, 72, 74
    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


  • Here are the quantiluore results from the WP-PCR of 0.1 nM ΦX174 with variable cycle number, N.
    1. 0 - nM ( %)
    2. 5 - nM ( %)
    3. 10 - nM ( %)
    4. 15 - nM ( %)
    5. 20 - nM ( %)
    6. 25 - nM ( %)
    7. 30 - nM ( %)
    8. 35 - nM ( %)
    9. 40 - nM ( %)
  • Optimizing N, the number of PCR cycles. 50 μL + 10% WP-PCR reaction:
    • 34.3 μL H2O
    • 6.88 μL 2mM dNTPs (each) (0.25mM each final)
    • 5.5 μL 10X reaction buffer
    • 5.5 μL ??? μM primer 4 mix
    • 1.719 μL 3.2 nM ΦX174 template (0.1 nM final)
    • 1.1 μL PfuUltra II fusion HS DNA polymerase
  • Aliquot 5×10μL and repeat 5 WP-PCR reactions for variable Ta, where Ta = 54, 56, 58, 60, and 62 °C.
  • Cycling parameters:
    1. 95 °C 2 min
    2. 95 °C 20 s
    3. Ta °C 20 s
    4. 72 °C ??? s
    5. Repeat 2-4 an additional ??? times = ??? cycles
    6. 72 °C 3 min
    7. 12 °C hold
  • Next on the list:
    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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