User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/18: Difference between revisions
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* Gel results from WP-PCR on 09/17/2012 showed no amplification. Discussed with Vincent. We will go over my protocols and results in the lab meeting. | * Gel results from WP-PCR on 09/17/2012 showed no amplification. Discussed with Vincent. We will go over my protocols and results in the lab meeting. | ||
* I checked the PCR program and noticed I used 68 °C for elongation, instead of 72 °C like I thought. | |||
* One more try. This time I will use 10 μM primer mix, which worked previously. | |||
* 25 μL WP-PCR reaction: | |||
** 31.2 μL H<sub>2</sub>O | |||
** 3.12 μL 2mM dNTPs (each) (0.25mM each final) | |||
** 2.5 μL 10X reaction buffer | |||
** 2.5 μL 10 μM primer 4 mix | |||
** 0.78 μL 3.2 nM ΦX174 template (0.1 nM final) | |||
** 0.5 μL PfuUltra I DNA polymerase | |||
* Cycling parameters: | |||
*# 95 °C 2 min | |||
*# 95 °C 30 s | |||
*# 58° C 30 s | |||
*# 72 °C 12 min | |||
*# Repeat 2-4 an additional 29 times for 30 total cycles | |||
*# 72 °C 30 min | |||
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Revision as of 15:34, 18 September 2012
PHIX174 Cell Free Expression | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
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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