User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/26

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m (Hypothesis 2: Gene L is necessary for phage propagation.)
Current revision (14:29, 28 September 2012) (view source)
m (Hypothesis 2: Gene L is necessary for phage propagation.)
 
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** 0.5 μL 25 mM dNTPs mix
** 0.5 μL 25 mM dNTPs mix
** 1 μL PfuUltra I DNA polymerase
** 1 μL PfuUltra I DNA polymerase
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* Next on the list:
 
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*# DpnI digestion (w/ and w/o PCR purification)
 
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*# Topisomerase IV / Gyrase (preceded by PCR purification) - how to assay linking number?
 
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* 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
 
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** experimental 1 = +template, +primer 4, +DNAP
 
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** experimental 2 = +template. +primer 4 T3585A, +DNAP
 
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** control 1: -template, +primer 4, +DNAP
 
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** control 2: -template, +primer 4 T3485, +DNAP
 
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** 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.===

Current revision

PHIX174 Cell Free Expression Main project page
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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 H2O
    • 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.

  • Re-concentrated pBEST-Pr-MG-apt-UTR1-deGFP-T500. Quantifluore measured concentration to be 231.5 nM (1.6%).
  • Repeat cell-free expression, now with top plasmid concentration point actually at 10. Also, do it at gain range of 200, 225, and 250.
  • Cell-free expression 80%:
    • 30 μL extract (12May11)
    • 1.17 μL water
    • 0.9 μL 100 mM Mg-glutamate (1 mM final)
    • 2 μL 3 M K-glutamate (66.7 mM final)
    • 22.5 μL 6 mM amino acids mix (1.5 mM final)
    • 6.43 μL 14X 3-PGC buffer (1X final)
    • 4.5 μL PEG8000 40% (2% final)
    • 4.5 μL 200 μM malachite green (10 μM final)
  • Aliquot 6 × 8 μL into 394-well plate and then add plasmid (pBEST-Pr-MGapt-UTR1-deGFP-T500) range:
    1. 2 μL water = 0 nM
    2. 2 μL 50 nM / 3^4 * 20% = .123 nM
    3. 2 μL 50 nM / 3^3 * 20% = .370 nM
    4. 2 μL 50 nM / 3^2 * 20% = 1.11 nM
    5. 2 μL 50 nM / 3^1 * 20% = 3.33 nM
    6. 2 μL 50 nM / 3^0 * 20% = 10 nM
  • Plate reader:
    1. 29 °C
    2. Shake: fast double orbital 30s
    3. Read: 625/655 nM @ gain = 200 w/ settings
    4. Read: 625/655 nM @ gain = 225 w/ settings
    5. Read: 625/655 nM @ gain = 250 w/ settings
    6. Loop: 2-5 every 3 m for 12 h
    • settings = full plate, high lamp energy, endpoint, bottom reading, other settings default
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