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 ~14.5 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:
- Cycling parameters:
- 95 °C 2 min
- 95 °C 20 s
- 58 °C 20 s
- X °C 12 min, , where X = 66, 68, 70, 72, 74
- Repeat 2-4 an additional 29 times = 30 cycles
- 12 °C hold
- Publication idea: describe preparation of cell free expression template by WP-PCR. Include WP-PCR optimization, ligation, and gyrase studies. Estimate the mutated population of PCR products using The Polymerase Chain Reaction and Branching Processes by F. Sun.
- 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).
- 66 °C - nM (%)
- 68 °C - nM (%)
- 70 °C - nM (%)
- 72 °C - nM (%)
- 74 °C - nM (%)
- Results ...
- Next, testing variable annealing temperature: Te = 54, 56, 58, 60, 62 °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:
- 95 °C 2 min
- 95 °C 20 s
- X °C 20 s, where X = 54, 56, 58, 60, 62
- X °C 12 min
- Repeat 2-4 an additional 29 times = 30 cycles
- 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 annealing temperature, Ta).
- 54 °C - nM (%)
- 56 °C - nM (%)
- 58 °C - nM (%)
- 60 °C - nM (%)
- 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:
- 95 °C 2 min
- 95 °C 20 s
- X° C 20 s
- X °C 12 min
- 25 °C hold
- 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
- 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.
- 0 - nM (%)
- 5 - nM (%)
- 10 - nM (%)
- 15 - nM (%)
- 20 - nM (%)
- 25 - nM (%)
- 30 - nM (%)
- 35 - nM (%)
- 40 - nM (%)
- 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:
- 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:
- Ta = 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, 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.
- 0 - nM ( %)
- 5 - nM ( %)
- 10 - nM ( %)
- 15 - nM ( %)
- 20 - nM ( %)
- 25 - nM ( %)
- 30 - nM ( %)
- 35 - nM ( %)
- 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:
- 95 °C 2 min
- 95 °C 20 s
- Ta °C 20 s
- 72 °C ??? s
- Repeat 2-4 an additional ??? times = ??? cycles
- 72 °C 3 min
- 12 °C hold
- Next on the list:
- 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, 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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