User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/18: Difference between revisions

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* 25 μL WP-PCR reaction:
* 25 μL WP-PCR reaction:
** 31.2 μL H<sub>2</sub>O
** 15.6 μL H<sub>2</sub>O
** 3.12 μL 2mM dNTPs (each) (0.25mM each final)
** 3.12 μL 2mM dNTPs (each) (0.25mM each final)
** 2.5 μL 10X reaction buffer
** 2.5 μL 10X reaction buffer

Revision as of 16:16, 18 September 2012

PHIX174 Cell Free Expression <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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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 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:
    • 15.6 μL H2O
    • 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:
    1. 95 °C 2 min
    2. 95 °C 30 s
    3. 58° C 30 s
    4. 72 °C 12 min
    5. Repeat 2-4 an additional 29 times for 30 total cycles
    6. 72 °C 30 min
  • Next on the list:
    1. DpnI digestion (w/ and w/o PCR purification)
    2. 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, Ta, 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

Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.

  • Many problems experienced over the summer regarding construction of PX-UTR1-deGFP and PX-UTRX-deGFP.
  • Transformed pBEST-Pr-MGapt-UTR1-deGFP-T500 into KL740.
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