User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/27: Difference between revisions
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===Hypothesis 2: Gene L is necessary for phage propagation.=== | |||
* Last time, I verified the final WP-PCR protocol. | |||
* Now, I want to see if DpnI digestion works or not on the methylated ΦX174 template genomic DNA in the PCR conditions. | |||
* 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. | |||
* 50 μL WP-PCR reaction w/o primers: | |||
** 37 μL H<sub>2</sub>O | |||
** 7.5 μL 100 nM ΦX174 template (~15 nM final) | |||
** 5 μL 10X reaction buffer | |||
** 0.5 μL 25 mM dNTPs mix | |||
** 1 μL PfuUltra I DNA polymerase | |||
* WP-PCR Cycling parameters: | |||
*# 95 °C 2 m | |||
*# 95 °C 30 s | |||
*# 58° C 30 s | |||
*# 72 °C 15 m | |||
*# Repeat 2-4 an additional 29 times for 30 total cycles | |||
*# 72 °C 30 m | |||
* I prepared this today (Fri) and placed in the Chromato. Vincent will run the WP-PCR Mon. morning. | |||
----------------- | |||
* Next on the list: | |||
*# 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 | |||
===Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.=== | ===Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.=== | ||
Revision as of 11:44, 28 September 2012
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Hypothesis 2: Gene L is necessary for phage propagation.
Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.
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