Difference between revisions of "User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/28"

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(Autocreate 2012/09/28 Entry for User:Sean_P_Corum/Notebook/PHIX174_Cell_Free)
 
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==Entry title==
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===Hypothesis 2: Gene L is necessary for phage propagation.===
* Insert content here...
 
  
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* Last time, I verified the final WP-PCR protocol.
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* Now, I want to see if DpnI digestion works or not on the methylated ΦX174 template genomic DNA in the PCR conditions.
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* For there to be 0.5 μg of DNA to digest in 10 μL, which is enough to see on a gel, I'll need 50 ng/μL ~ 15 nM final concentration DNA.
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* 50 μL WP-PCR reaction w/o primers:
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** 37 μL H<sub>2</sub>O
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** 7.5 μL 100 nM ΦX174 template (~15 nM final)
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** 5 μL 10X reaction buffer
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** 0.5 μL 25 mM dNTPs mix
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** 1 μL PfuUltra I DNA polymerase
  
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__NOTOC__
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-----------------
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* Next on the list:
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*# DpnI digestion (w/ and w/o PCR purification)
 +
*# 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
 +
** experimental 1 = +template, +primer 4, +DNAP
 +
** 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

Revision as of 10:41, 28 September 2012

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Hypothesis 2: Gene L is necessary for phage propagation.

  • Last time, I verified the final WP-PCR protocol.
  • Now, I want to see if DpnI digestion works or not on the methylated ΦX174 template genomic DNA in the PCR conditions.
  • For there to be 0.5 μg of DNA to digest in 10 μL, which is enough to see on a gel, I'll need 50 ng/μL ~ 15 nM final concentration DNA.
  • 50 μL WP-PCR reaction w/o primers:
    • 37 μL H2O
    • 7.5 μL 100 nM ΦX174 template (~15 nM final)
    • 5 μL 10X reaction buffer
    • 0.5 μL 25 mM dNTPs mix
    • 1 μL PfuUltra I DNA polymerase



  • 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