User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/08/21: Difference between revisions

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* The last three data points satisfies the linear equation product = 4.99x+67.5 with R<sup>2</sup> = 0.9997. The fact that this is growing linear indicates that the primers are the limiting component in WP-PCR (as primers typically are in WP-PCR). Next, I will optimize elongation time.
* The last three data points satisfies the linear equation product = 4.99x+67.5 with R<sup>2</sup> = 0.9997. The fact that this is growing linear indicates that the primers are the limiting component in WP-PCR (as primers typically are in WP-PCR). Next, I will optimize elongation time.


* Before this, I re-measured "100" μM S and AS primers by quanitfluore
* Before this, I measured the stock "1" mM S and AS primers by quanitfluore
*# "100" μM S primer 4 -  μM
*# "1" mM S primer 4 -  mM
*# "100" μM AS primer 4v -  μM
*# "1" mM AS primer 4v -  mM


* Given these results, the 1 mM primer stocks were actually:
* Given these results, I made more accurate stocks of S and AS primer 4:
*# 1 mM S primer 4 →
** S:
*# 1 mM AS primer 4 →
** AS:


* Optimize elongation time. 50 μL WP-PCR reaction:
* Optimize elongation time. 50 μL WP-PCR reaction:

Revision as of 16:07, 21 August 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.

  • Here is the quantiluore results from the WP-PCR of 0.1 nM ΦX174 with variable 36-mer primer concentration. ΦX174 1 nM template only control gave ~0 nM w/ inconsistent readings (not really a problem).
    1. 0 μM - 0.36 nM inconsistent
    2. 1 μM - 0.31 nM inconsistent
    3. 2 μM - 78 nM
    4. 5 μM - 92 nM
    5. 10 μM - 117 nM
    • 100 μM S primer - 39 μM (0.8%)
    • 100 μM AS primer - 60 μM (18%); this needs to be repeated
  • The last three data points satisfies the linear equation product = 4.99x+67.5 with R2 = 0.9997. The fact that this is growing linear indicates that the primers are the limiting component in WP-PCR (as primers typically are in WP-PCR). Next, I will optimize elongation time.
  • Before this, I measured the stock "1" mM S and AS primers by quanitfluore
    1. "1" mM S primer 4 - mM
    2. "1" mM AS primer 4v - mM
  • Given these results, I made more accurate stocks of S and AS primer 4:
    • S:
    • AS:
  • Optimize elongation time. 50 μL WP-PCR reaction:
    • 29.6 μL H2O
    • 5.5 μL 10X reaction buffer
    • 6.88 μL 2mM dNTPs (each) (0.25mM each final)
    • 5.5 μL primer 4 mix (each)
    • 1.562 μL 3.2 nM ΦX174 template (0.1nM final)
    • 1 μL PfuUltra II fusion HS DNA polymerase
  • Aliquot 5×10 μL.
  • Cycling parameters:
    1. 95 °C 2 min
    2. 95 °C 20 s
    3. 58 °C 20 s
    4. 72 °C X min, where X 0.5, 1, 2, 5, or 10
    5. Repeat 2-4 an additional 29 times = 30 cycles
    6. 72 °C 3 min
    7. 12 °C hold
  • Things that still need to be optimized:
    1. After primer concentrations have been corrected, vary primer concentration by 0, 1E2, 1E3, 1E4, 1E5, 1E6 nM = 1 mM
    2. Ta = 54, 56, 58, 60, 62 °C
  • N cycles = 0, 10, 20, 25, 30, 35, 40, 50
  • After that, tasks include characterizing effects of:
    1. parallel PFU ligation
    2. DpnI digestion (w/ and w/o PCR purification)
    3. 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
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