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:
** 
** 0.5 μM: stong ~≥ 5 kb (amplified ΦX174 genome), weak ~>100 bp (band 2)
** 0.5 μM: stong ~≥ 5 kb (amplified ΦX174 genome), weak ~>100 bp (band 2)
** 0.5 μM: stong ~≥ 5 kb (amplified ΦX174 genome), weak ~>100 bp (band 2)
** 0.5 μM: stong ~≥ 5 kb (amplified ΦX174 genome), weak ~>100 bp (band 2)
** 0.5 μM: stong ~≥ 5 kb (amplified ΦX174 genome), weak ~>100 bp (band 2)
** 0.5 μM: stong ~≥ 5 kb (amplified ΦX174 genome), weak ~>100 bp (band 2)
* Given these results, here's what I think is happening. At too high primer concentration, the ssDNA primers, being complementary strands of WP-PCR, do not unbind, inhibiting PCR.
* I will do one more test over a primer 4 mix of 0, 0.5, 1, 2, 4, and 8 μM.
* Aliquot 6 × 4.5 μL ΦX174 WP-PCR mix (1X reaction buffer, .25 mM dNTPs mix, PfuUltra I DNAP, ~0.1 nM ΦX174 template)
*# 0.5 μL 5 water
*# 0.5 μL 5 μM primer 4 mix
*# 0.5 μL 10 μM primer 4 mix
*# 0.5 μL 20 μM primer 4 mix
*# 0.5 μL 40 μM primer 4 mix
*# 0.5 μL 80 μM primer 4 mix
* WP-PCR Cycling parameters:
*# 95 °C 2 m
*# 95 °C 30 s
*# 58° C 30 s
*# 72 °C 15 m
*# Repeat 2-4 an additional 29 times for 30 total cycles
*# 72 °C 30 m
-----------------
* Next on the list:
*# DpnI digestion (w/ and w/o PCR purification)
*# 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, T<sub>a</sub>, 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
===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 13:41, 27 September 2012

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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:
    • 0.5 μM: stong ~≥ 5 kb (amplified ΦX174 genome), weak ~>100 bp (band 2)
    • 0.5 μM: stong ~≥ 5 kb (amplified ΦX174 genome), weak ~>100 bp (band 2)
    • 0.5 μM: stong ~≥ 5 kb (amplified ΦX174 genome), weak ~>100 bp (band 2)
    • 0.5 μM: stong ~≥ 5 kb (amplified ΦX174 genome), weak ~>100 bp (band 2)
    • 0.5 μM: stong ~≥ 5 kb (amplified ΦX174 genome), weak ~>100 bp (band 2)
    • 0.5 μM: stong ~≥ 5 kb (amplified ΦX174 genome), weak ~>100 bp (band 2)
  • Given these results, here's what I think is happening. At too high primer concentration, the ssDNA primers, being complementary strands of WP-PCR, do not unbind, inhibiting PCR.
  • I will do one more test over a primer 4 mix of 0, 0.5, 1, 2, 4, and 8 μM.
  • Aliquot 6 × 4.5 μL ΦX174 WP-PCR mix (1X reaction buffer, .25 mM dNTPs mix, PfuUltra I DNAP, ~0.1 nM ΦX174 template)
    1. 0.5 μL 5 water
    2. 0.5 μL 5 μM primer 4 mix
    3. 0.5 μL 10 μM primer 4 mix
    4. 0.5 μL 20 μM primer 4 mix
    5. 0.5 μL 40 μM primer 4 mix
    6. 0.5 μL 80 μM primer 4 mix
  • WP-PCR Cycling parameters:
    1. 95 °C 2 m
    2. 95 °C 30 s
    3. 58° C 30 s
    4. 72 °C 15 m
    5. Repeat 2-4 an additional 29 times for 30 total cycles
    6. 72 °C 30 m


  • 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


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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