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| |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> PHIX174 Cell Free Expression</span>
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| |style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html> </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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| <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
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| ===Hypothesis 2: Gene L is necessary for phage propagation.===
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| * Here is the quantiluore results from the WP-PCR of 0.1 nM ΦX174 with variable elongation time. Time is is min:sec:
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| *# 10min/3^4 = 00:07 - nM ( %)
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| *# 10min/3^3 = 00:22 - nM ( %)
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| *# 10min/3^2 = 01:07 - nM ( %)
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| *# 10min/3^1 = 03:20 - nM ( %)
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| *# 10min/3^0 = 10:00 - nM ( %)
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| *# "0.1" nM ΦX174 - nM ( %)
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| * The last three data points satisfies the linear equation product = 4.99x+67.5 with R<sup>2</sup> = 0.9997. The fact that this is growing linear indicates that the primers are the limiting component in WP-PCR (as primers typically are in WP-PCR).
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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:
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| ** 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:
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| ** (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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