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| - | ===Hypothesis 2: Gene L is necessary for phage propagation.===
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| - | * Last time, I verified the final WP-PCR protocol.
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| - | * Now, I want to see if DpnI digestion works or not on the methylated ΦX174 template genomic DNA in the PCR conditions.
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| - | * For there to be 0.5 μg of DNA to digest in 10 μL, which is enough to see on a gel, I'll need 50 ng/μL ~ 15 nM final concentration DNA.
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| - | * 50 μL WP-PCR reaction w/o primers:
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| - | ** 37 μL H<sub>2</sub>O
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| - | ** 7.5 μL 100 nM ΦX174 template (~15 nM final)
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| - | ** 5 μL 10X reaction buffer
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| - | ** 0.5 μL 25 mM dNTPs mix
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| - | ** 1 μL PfuUltra I DNA polymerase
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| - |
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| - |
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| - | -----------------
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| - |
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| - | * Next on the list:
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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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PHIX174 Cell Free Expression
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