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| Line 6: |
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| ===Hypothesis 2: Gene L is necessary for phage propagation.===
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|
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| * 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:
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| *# 0:00 - 4.8 nM (1.3%)
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| *# 1:00 - 8.6 nM (0.4%)
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| *# 2:00 - 20.4 nM (1.0%)
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| *# 3:00 - 30.6 nM (0.04%)
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| *# 4:00 - 27.8 nM (2%)
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| *# 5:00 - 44.3 nM (4%)
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| *# 10:00 - 47.9 nM (2%)
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| * 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.
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| * Next, testing variable elongation temperature: T<sup>e</sup> = 66, 68, 70, 72, 74 °C
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| * 50 μL WP-PCR:
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| ** 30.2 μL H<sub>2</sub>O
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| ** 6.25 μL 2mM dNTPs (each) (0.25mM each final)
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| ** 5.5 μL 10X reaction buffer
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| ** 5.5 μL 10 μM primer 4 mix
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| ** 1.563 μL 3.2 nM ΦX174 template (0.1 nM final)
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| ** 1 μL PfuUltra II fusion HS DNA polymerase
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| * Aliquot 5×10μL.
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| * Cycling parameters:
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| *# 95 °C 2 min
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| *# 95 °C 20 s
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| *# 58 °C 20 s
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| *# X °C 10 min, where X = 66, 68, 70, 72, 74
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| *# Repeat 2-4 an additional 29 times = 30 cycles
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| *# 12 °C hold
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|
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| -------------------
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|
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| * 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
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| ** 6.6 μL 10X reaction buffer
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| ** 8.25 μL 2mM dNTPs (each) (0.25mM each final)
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| ** 2.063 μL 3.2 nM ΦX174 template (0.1 nM final)
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| ** 1.32 μL PfuUltra II fusion HS DNA polymerase
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| * Aliquot 6×9 μL and add 10X primer range (0, 100 nM, 1 μM, 10 μM, 100 μM, and 1 mM).
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| * Cycling parameters:
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| *# 95 °C 2 min
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| *# 95 °C 20 s
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| *# 58 °C 20 s
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| *# 72 °C ??? s
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| *# Repeat 2-4 an additional 29 times = 30 cycles
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| *# 72 °C 3 min
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| *# 12 °C hold
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| * Things that still need to be optimized:
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| *# T<sub>a</sub> = 54, 56, 58, 60, 62 °C
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| *# N cycles = 0, 10, 20, 25, 30, 35, 40, 50
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| * After that, tasks include characterizing effects of:
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| *# parallel PFU ligation
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| *# DpnI digestion (w/ and w/o PCR purification)
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| *# Topisomerase IV / Gyrase (preceded by PCR purification) - how to assay linking number?
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| * 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
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| ** experimental 1 = +template, +primer 4, +DNAP
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| ** experimental 2 = +template. +primer 4 T3585A, +DNAP
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| ** control 1: -template, +primer 4, +DNAP
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| ** control 2: -template, +primer 4 T3485, +DNAP
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| ** control 3: +template, +primer 4, +DNAP
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| ** control 4: +template, +primer 4 T3485, +DNAP
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| ** control 5: +template, -primers, +DNAP
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| * Followed by:
| |
| ** (possible purification and then) DpnI digestion
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| ** PCR purification
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| ** Linking number adjustment by gyrase / topisomerase IV
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| ** PCR purification
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|
| |
|
| |
| * Here are the quantiluore results from the WP-PCR of 0.1 nM ΦX174 with variable cycle number, N.
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| *# 0 - nM ( %)
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| *# 5 - nM ( %)
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| *# 10 - nM ( %)
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| *# 15 - nM ( %)
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| *# 20 - nM ( %)
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| *# 25 - nM ( %)
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| *# 30 - nM ( %)
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| *# 35 - nM ( %)
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| *# 40 - nM ( %)
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|
| |
| * Optimizing N, the number of PCR cycles. 50 μL + 10% WP-PCR reaction:
| |
| ** 34.3 μL H<sub>2</sub>O
| |
| ** 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 T<sub>a</sub>, where T<sub>a</sub> = 54, 56, 58, 60, and 62 °C.
| |
| * Cycling parameters:
| |
| *# 95 °C 2 min
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| *# 95 °C 20 s
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| *# T<sub>a</sub> °C 20 s
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| *# 72 °C ??? s
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| *# Repeat 2-4 an additional ??? times = ??? cycles
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| *# 72 °C 3 min
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| *# 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, 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
| |
PHIX174 Cell Free Expression
