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oxfordjuly072008:start [2014/05/15 17:34] – magierooxfordjuly072008:start [2015/01/30 21:19] (current) – magiero
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 ==== - Molecular Adaptor ==== ==== - Molecular Adaptor ====
 +
 +•[0141] …molecular adaptor…facilitates the interaction between the pore and the nucleotides.  The adaptor typically alters the charge of the barrel or channel of the pore or specifically interacts with or binds to nucleotides \\
 +•[0142] The adaptor preferably constricts the barrel or channel so that it may interact with the nucleotides. \\
 +•[0143] The adaptor is typically cyclic.  The adaptor preferably has the same symmetry as the pore.
  
 ==== - Covalent Attachment ==== ==== - Covalent Attachment ====
 +
 +• [0149] The adaptor is covalently attached to the pore.\\
 +• [0150] The site of covalent attachment is selected such that the adaptor is positioned at or near residue 139 of SEQ ID NO:2. **(This denotes the vertical position of the adaptor)**\\
  
 ==== - Positioning of the Adaptor ==== ==== - Positioning of the Adaptor ====
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 ==== - Apparatus ==== ==== - Apparatus ====
-• [0197] An example apparatus consists of two chambers separated by the pore carrying membrane.+• [0197] An example apparatus consists of two chambers separated by the pore carrying membrane.\\
 • [0199] Voltage clamp is preferred to patch clamp. **What is the reason for this preference?** • [0199] Voltage clamp is preferred to patch clamp. **What is the reason for this preference?**
  
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 ==== - Exonuclease ==== ==== - Exonuclease ====
-• [0212] Attach an exonuclease to the pore.  An exonuclease is a sequence snipping enzyme is processes DNA like a ribbon sequentially chopping off (**digesting**) nucleotides one after another.\\+• [0212] Attach an exonuclease to the pore.  An exonuclease is a sequence snipping enzyme. It processes DNA like a ribbon sequentially chopping off (**digesting**) nucleotides one after another.\\
 • [0215] Preferable to covalently attach the exonuclease to the pore.\\ • [0215] Preferable to covalently attach the exonuclease to the pore.\\
 • [0216] Preferable exonuclease rates are 1, 10, 100, 500, 1000 nucleotides per second.\\ • [0216] Preferable exonuclease rates are 1, 10, 100, 500, 1000 nucleotides per second.\\
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 === - Data Analysis and Acquisition === === - Data Analysis and Acquisition ===
-• [0236] Axopatch 200B patch clamp amplifier used.  4-pole 10-kHz Bessel filter.  Sampled at 20 kHz by PC equipped with Digidata 1440A A/D (Axon instruments) running CampEx 10 software (Molecular Devices).\\+• [0236] Axopatch 200B patch clamp amplifier used.  4-pole 10-kHz Bessel filter.  Sampled at 20 kHz by PC equipped with Digidata 1440A A/D (Axon instruments) running ClampEx 10 software (Molecular Devices).\\
 • [0237] Event histograms are constructed as follows…\\ • [0237] Event histograms are constructed as follows…\\
 • [0238] Run statistics on a moving window consisting of WT points.\\ • [0238] Run statistics on a moving window consisting of WT points.\\
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 === - High Positions of Attachment--Residues 115 to 121 === === - High Positions of Attachment--Residues 115 to 121 ===
  
-• [0261] In the 115 position in <m>\beta</m>-barrel baseline was too noise and showed large current fluctuations.  Maybe due to movement of cyclodextrin in barrel.\\+• [0261] In the 115 position in the <m>\beta</m>-barrel the baseline was too noisy and showed large current fluctuations.  Maybe due to movement of cyclodextrin in barrel.\\
 • [0263] Further down in 119 position baseline got cleaner, but still showed some spikes.\\ • [0263] Further down in 119 position baseline got cleaner, but still showed some spikes.\\
  
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 • [0280] As you increase the applied voltage you get increase in passing currents and not necessarily equal shifts in the histograms (Fig. 18).\\ • [0280] As you increase the applied voltage you get increase in passing currents and not necessarily equal shifts in the histograms (Fig. 18).\\
 • [0281] Histogram overlab (at different voltages) looks pretty small (Fig. 19).\\ • [0281] Histogram overlab (at different voltages) looks pretty small (Fig. 19).\\
-• [0282] The dTMP histogram looks the sharpest.  Why?+• [0282] The dTMP histogram looks the sharpest.  Why?\\
 • [0283] Because its **dwell time** is the largest.  The longer (in time) the signal it generates the more samples we accrue and hence the more accurate our sampling of it (therefore the sharper the histogram). A summary of avg. dwell times in Fig. 20 at 130 mV and 800 mM KCl (just eyeballed the max. dwell times from Fig. 20). • [0283] Because its **dwell time** is the largest.  The longer (in time) the signal it generates the more samples we accrue and hence the more accurate our sampling of it (therefore the sharper the histogram). A summary of avg. dwell times in Fig. 20 at 130 mV and 800 mM KCl (just eyeballed the max. dwell times from Fig. 20).
  
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 | dCMP    |  7.3 ms        |  48 ms          | | dCMP    |  7.3 ms        |  48 ms          |
  
-**Recall**, sampling time is 20-kHz so every 0.05 ms.  So on average they are **oversampling** by about 100 to 400 times?\\+**Recall**, sampling time is 20-kHz so every 0.05 ms.  Avg. dwell time is 9.4 ms, so on average they are **oversampling** by 188 times.\\
  
 === - Low Salt Operating Conditions === === - Low Salt Operating Conditions ===
oxfordjuly072008/start.1400175275.txt.gz · Last modified: 2014/05/15 17:34 by magiero
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