User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/26: Difference between revisions
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* Therefore, as I anticipated, too much primers inhibited PCR due to primer hybridization of complementary primers used for WP-PCR. | * Therefore, as I anticipated, too much primers inhibited PCR due to primer hybridization of complementary primers used for WP-PCR. | ||
* 0.5 μM should be used for WP-PCR amplification of ΦX174 genome. I finalized the WP-PCR protocol. Other long WP-PCRs should use between 0.1 and 1 μM primer, with the optimal primer concentration being found for each new primer/template combination. | * 0.5 μM should be used for WP-PCR amplification of ΦX174 genome. I finalized the WP-PCR protocol. Other long WP-PCRs should use between 0.1 and 1 μM primer, with the optimal primer concentration being found for each new primer/template combination. | ||
* Final WP-PCR protocol | * Final WP-PCR protocol is ... | ||
* 50 μL WP-PCR reaction: | |||
* | ** 37 μL H<sub>2</sub>O | ||
** | ** 5 μL 10X reaction buffer | ||
** 5 μL 5 μM primer mix (0.5 μM final, each) | |||
** | ** 1.5 μL 3.2 nM ΦX174 template (~ 0.1 nM final) | ||
** | ** 0.5 μL 25 mM dNTPs mix | ||
** 1 μL PfuUltra I DNA polymerase | |||
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** control 2: -template, +primer 4 T3485, +DNAP | ** control 2: -template, +primer 4 T3485, +DNAP | ||
** control 3: +template, +primer 4, +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 13:43, 27 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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