User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/26: Difference between revisions
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===Hypothesis 2: Gene L is necessary for phage propagation.=== | |||
* Gel electrophoresis showed the following over the rest of the primer 4 mix range: | |||
** Nothing the the column of -primer control. | |||
** A strong band at > 5kb at 0.5 μM primer (band 1), growing weaker as primer concentration increased, and disappearing for the 4 and 8 μM primer columns. Band 1 = amplified, nicked ΦX174 genome. | |||
** A weak band at < 100 bp (band 2) at 0.5 μM primer (band 2), growing stronger as primer concentration increased. Band 2 = ssDNA primers. | |||
** A weak band at < 100 bp (band 3) > band 2, not apparent until 1 μM (nothing at 0.5 μM) and growing stronger with increasing primer concentration. Band 3: hybridized ssDNA primers. | |||
* 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. | |||
* 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 | |||
===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 11:29, 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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