User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/18: Difference between revisions
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== | ===Hypothesis 2: Gene L is necessary for phage propagation.=== | ||
* Gel results from WP-PCR on 09/17/2012 showed no amplification. Discussed with Vincent. We will go over my protocols in the lab meeting. | |||
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* Next on the list: | |||
*# 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 | |||
Revision as of 14:19, 18 September 2012
 PHIX174 Cell Free Expression
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Hypothesis 2: Gene L is necessary for phage propagation.
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