User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/08/29: Difference between revisions

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* Results show maximal PCR at 72 °C, as expected. I recall the only reason I checked this is that the included technical reference for the PfuUltra II Fusion DNAP was incorrect on elongation time.
* Results show maximal PCR at 72 °C, as expected. I recall the only reason I checked this is that the included technical reference for the PfuUltra II Fusion DNAP was incorrect on elongation time.
* Also performed gel electrophoresis on the samples. 0.1 nM ΦX174 showed no visible band, while the PCRed samples showed a distinct band at ~≥ 5 kb linear dsDNA, which is expected, since the samples were 5.4 kb nicked. This is reassuring, since this is the first time I actually viewed good results of ΦX174 WP-PCR.
* Also performed gel electrophoresis on the samples. 0.1 nM ΦX174 showed no visible band, while the PCRed samples showed a distinct band at ~≥ 5 kb linear dsDNA, which is expected, since the samples were 5.4 kb nicked. This is reassuring, since this is the first time I actually viewed good results of ΦX174 WP-PCR.
* Next, testing variable annealing temperature: T<sup>e</sup> = 54, 56, 58, 60, 62 °C
* Next, testing variable annealing temperature: T<sub>a</sub> = 54, 56, 58, 60, 62 °C
* 50 μL WP-PCR:
* 50 μL WP-PCR:
** 31.2 μL H<sub>2</sub>O
** 31.2 μL H<sub>2</sub>O
Line 30: Line 30:
*# 95 °C 20 s
*# 95 °C 20 s
*# X °C 20 s, where X = 54, 56, 58, 60, 62  
*# X °C 20 s, where X = 54, 56, 58, 60, 62  
*# X °C 2 min (this is suboptimal on purpose, in order to get though the experiment more quickly)
*# 72 °C 2 min (this is suboptimal on purpose, in order to get though the experiment more quickly)
*# Repeat 2-4 an additional 29 times = 30 cycles
*# Repeat 2-4 an additional 29 times = 30 cycles
*# 12 °C hold
*# 12 °C hold

Revision as of 17:49, 29 August 2012

PHIX174 Cell Free Expression <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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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 temperature, Te).
    1. 66 °C - 5.9 nM (2%)
    2. 68 °C - 10.4 nM (20%)
    3. 70 °C - 19.3 nM (15%)
    4. 72 °C - 49.4 nM (7%)
    5. 74 °C - 43.1 nM (6%)
  • Results show maximal PCR at 72 °C, as expected. I recall the only reason I checked this is that the included technical reference for the PfuUltra II Fusion DNAP was incorrect on elongation time.
  • Also performed gel electrophoresis on the samples. 0.1 nM ΦX174 showed no visible band, while the PCRed samples showed a distinct band at ~≥ 5 kb linear dsDNA, which is expected, since the samples were 5.4 kb nicked. This is reassuring, since this is the first time I actually viewed good results of ΦX174 WP-PCR.
  • Next, testing variable annealing temperature: Ta = 54, 56, 58, 60, 62 °C
  • 50 μL WP-PCR:
    • 31.2 μL H2O
    • 6.25 μL 2mM dNTPs (each) (0.25mM each final)
    • 5 μL 10X reaction buffer
    • 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. X °C 20 s, where X = 54, 56, 58, 60, 62
    4. 72 °C 2 min (this is suboptimal on purpose, in order to get though the experiment more quickly)
    5. Repeat 2-4 an additional 29 times = 30 cycles
    6. 12 °C hold
  • Here is the quantiluore results from the WP-PCR of 0.1 nM ΦX174 and 1 μMM primer 4 (each) with variable Ta).
    1. 54 °C - nM (%)
    2. 56 °C - nM (%)
    3. 58 °C - nM (%)
    4. 60 °C - nM (%)
    5. 62 °C - nM (%)
  • Results ...
  • Next, testing variable PCR cycling number N
  • 100 μL WP-PCR:
    • 62.4 μL H2O
    • 12.5 μL 2mM dNTPs (each) (0.25mM each final)
    • 10 μL 10X reaction buffer
    • 10 μL 10 μM primer 4 mix
    • 3.13 μL 3.2 nM ΦX174 template (0.1 nM final)
    • 2 μL PfuUltra II fusion HS DNA polymerase
  • Cycling parameters:
    1. 95 °C 2 min
    2. 95 °C 20 s
    3. X° C 20 s
    4. X °C 12 min
    5. 25 °C hold
    6. Repeat 2-5 an additional 44 times for N = 45 cycles, removing 10 μL samples for N = 0, 5, 10, 15, 20, 25, 30, 35, 40, 45
    7. 12 °C hold
  • Here is the quantiluore results from the WP-PCR of 0.1 nM ΦX174 and 1 μM primer 4 (each) with variable PCR 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 (%)
    10. 45 - nM (%)
  • Results ...


  • 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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