Biomod/2011/TUM/TNT/Results: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
No edit summary
 
(14 intermediate revisions by 4 users not shown)
Line 6: Line 6:


<h1>Survey of Results</h1>
<h1>Survey of Results</h1>
We successfully designed a structure that folds properly with high yields [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#Folding_.26_Purification (link)] and is suitable for observing the structural deformations. Comprehensive TEM analysis yielded insights into global structural deformations and allowed for statistical evaluation of angle and length distributions dependent on DNA binder concentrations [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#TEM_Image_Analysis (link)]. Our structure could be labeled with fluorescent dyes and a huge variety of different approaches to fluorescence measurements was tested. In single molecule measurements FRET events could be observed. [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#Fluorescence_Measurements (link)]. Based on these experimental data and also our structure simulations and calculations, we gained new insights into the structural properties of DNA origamis especially with regards to binding of small molecules [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#Discussion (link)].  
We successfully designed a structure that folds properly with high yields [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#Folding_.26_Purification (link)] and is suitable for observing structural deformations. Comprehensive TEM analysis yielded insights into global structural deformations and allowed for statistical evaluation of angle and length distributions dependent on DNA binder concentrations [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#TEM_Image_Analysis (link)]. Our structure was successfully labeled with fluorescent dyes and a considerable variety of different approaches to fluorescence measurements was tested. In single molecule measurements FRET events could be observed. [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#Fluorescence_Measurements (link)]. Based on these experimental data as well as our structure simulations and calculations, we gained new insights into the structural properties of DNA origamis especially with regards to binding of small molecules [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#Discussion (link)].  


<h1> Folding & Purification </h1>
<h1> Folding & Purification </h1>


<table cellspacing="0" cellpadding="2">
<table cellspacing="0" cellpadding="2">
<tr bgcolor="#f5f5f5"><td valign="top">The U structure was folded using the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Setting_up_folding_reactions 15_65] ramp. This ramp was the fastest of the tested ones and also led to proper folded origamis as shown in figure 1. There is only one major band visible in the agarose gel, indicating that no significant amounts of byproducts (like dimers) have been formed. The results of the slower ramps 2D_H3_ML and 5D_H3_ML yielded similar results as the 15_65 ramp. </td><td>[[Image:2011.08.24 theU 1kBlad-scaff-bm1-bm2.jpg|400x400px | thumb | Fig. 1 From left: 1kb-ladder, scaffold p7560, (scaffold p7560),  [[http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Structure_page#BM1_.26_BM2:_Unlabeled_structures BM1, BM2]]]]</td></tr>
<tr bgcolor="#f5f5f5"><td valign="top">The U structure was folded using the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Setting_up_folding_reactions 15_65] ramp. This ramp was the fastest of the tested ones and also led to properly folded origamis as shown in figure 1. There is only one major band visible in the agarose gel, indicating that no significant amounts of byproducts (like dimers) have been formed. The results of the slower ramps 2D_H3_ML and 5D_H3_ML yielded similar, not better, results as the 15_65 ramp, indicating an efficient folding even after such short time. </td><td>[[Image:2011.08.24 theU 1kBlad-scaff-bm1-bm2.jpg|400x400px | thumb | Fig. 1 From left: 1kb-ladder, scaffold p7560, (scaffold p7560),  [[http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Structure_page#BM1_.26_BM2:_Unlabeled_structures BM1, BM2]]]]</td></tr>
</table>
</table>




<table cellspacing="0" cellpadding="2">
<table cellspacing="0" cellpadding="2">
<tr bgcolor="#f5f5f5"><td valign="top">Most of the structures from the major band were folded correctly, which was proven by TEM images (figure 2).
<tr bgcolor="#f5f5f5"><td valign="top">Most of the structures from the major band were folded correctly, which was demonstrated by TEM imaging (figure 2).
The purification of the structures was tried both with an agarose gel and with an Amicon size exclusion filter (molecular weight cutoff: 100kDa). According to general experience, the yield of purification via agarose gel is approximately 2 nM. The yield of the filter purification was definitively higher, since unexpected weak dilutions led to appropriate concentrations for TEM and fluorescence microscope. Therefore we estimate the yield to be roughly 10 nM. </td><td>[[Image:20111021controltheUBM2 2B7.png|400px|thumb|Fig. 2 The U structure gallery (BM2_2B7)]]</td></tr>
The purification of the structures was tried both with an agarose gel and with an Amicon size exclusion filter (molecular weight cutoff: 100kDa). According to general experience, the yield of purification via agarose gel is approximately 2 nM. The yield of the filter purification was definitely higher, since even weak dilutions led to appropriate concentrations for TEM and fluorescence microscopy. Therefore we estimate the yield to be roughly 10 nM. </td><td>[[Image:20111021controltheUBM2 2B7.png|400px|thumb|Fig. 2 The U structure gallery (BM2_2B7)]]</td></tr>
</table>
</table>


Line 25: Line 25:
<h1>TEM Image Analysis</h1>
<h1>TEM Image Analysis</h1>
<h2>Distribution of Angles</h2>
<h2>Distribution of Angles</h2>
When we inspected the structure in the TEM, we saw a spread of the arms in the uprightly orientated structures (figure 3). The magnitude of this spread seemed to be correlated to the amount of DNA binding molecules.  
When we inspected the structure in the TEM, we saw a spread of the arms in the upright projections. (figure 3). The extent of this spread seemed to be correlated to the amount of DNA binding molecules.  


<table cellspacing="0" cellpadding="2">
<table cellspacing="0" cellpadding="2">
Line 38: Line 38:


<html>
<html>
<left><font size=1>Table 1: </font>
<center><font size=1><b>Table 1: Results from twist measurements</b></font></center>
<table cellspacing="0" cellpadding="6" align="center">
<table cellspacing="0" cellpadding="6" align="center">
<tr bgcolor="#f5f5f5">
<tr bgcolor="#f5f5f5">
<td><b></b></td><td><b>Number of particles</b></td><td><b>Mean angle [degree]</b></td></td><td><b>Variance</b></td></td></td>
<td></td><td><b>Number of particles</b></td><td><b>Mean angle [degree]</b></td></td><td><b>Variance</b></td></td></td>
</tr>
</tr>
<tr bgcolor="#f5f5f5">
<tr bgcolor="#f5f5f5">
Line 90: Line 90:
[[Image:DAPI 7 histo gaussian.png|390px]]
[[Image:DAPI 7 histo gaussian.png|390px]]


The distribution of angles in the control has two populations, one where the two arms are exactly above each other which leads to very small angles and one where the two arms are considerably spread. This leads to a gaussian distribution around an finite angle. The width of this distribution is in good agreement with the calculated [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Project#Thermal_Fluctuation_of_the_Arms], which yield deviations of ca. 4.4°. <br>
The distribution of angles in the negative control has two populations, one where the two arms are exactly above each other which leads to very small angles and one where the two arms are modestly spread. This leads to a gaussian distribution around a finite angle. The width of this distribution is in good agreement with the calculated [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Project#Thermal_Fluctuation_of_the_Arms thermal fluctuations], which yield deviations of approximately 4.4°. <br>


<table cellspacing="0" cellpadding="2">
<table cellspacing="0" cellpadding="2">
<tr bgcolor="#f5f5f5"><td valign="top">The positive control with an internally induced twist by additional base pairs in each helix (these additional base pairs lead to a net torque in each helix and therefore a macroscopic deformation of the structure) displays much higher angles. The population around zero is maybe due to deformed structures which had no second arm and could not be excluded. This results in many angles around zero. The other population around the finite angle is now the more spread structure. Here the angle of the positive control is shifted to higher values by approximately a factor of 2 because of the induced twist. So in principle this way of measuring the deformation of our structure in dependence of induced stress works.<br>The measured angles φ for negative and positive control, <math>\phi_{neg} \approx 9</math>° and <math>\phi_{pos} \approx 21</math>°, can be related to a torsion α of the base according to the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Project#Twist_of_the_Base theoretical considerations for the base twist]:
<tr bgcolor="#f5f5f5"><td valign="top">The positive control with an internally induced twist due to additional base pairs in each helix (these additional base pairs lead to a net torque in each helix and therefore a macroscopic deformation of the structure) displays much larger angles (see figure 5). The population around zero is probably caused by deformed structures which had no second arm but could not be visually excluded. This results in many angles around zero. The other population around the finite angle is now the more spread structure. Here the angle of the positive control is shifted to larger values by approximately a factor of 2 because of the induced twist. So in principle this way of measuring the deformation of our structure in dependence of induced stress works.<br>The measured angles φ for negative and positive control, <math>\phi_{neg} \approx 9</math>° and <math>\phi_{pos} \approx 21</math>°, can be related to a torsion α of the base according to the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Project#Twist_of_the_Base theoretical considerations for the base twist]:


<math>
<math>
Line 105: Line 105:


<h2>Length Measurements</h2>
<h2>Length Measurements</h2>
We also measured the lengths of the origami structures on the TEM images. Histograms of the length distributions display a gaussian shape (figure 6). For increasing concentrations of spermine, the length decreases steadily (figure 6a, for raw data see this file: <html><a href="http://openwetware.org/images/b/b6/TEM_length_measurements_raw_data.xlsx>TEM_length_measurements_raw_data.xlsx</a></html>. Surprisingly, for rising concentrations of ethidium bromide, the length decreases as well, although addition of ethidium bromide is known to increase the length of a simple double stranded DNA (figure 6b). It seems, that origami structures react here in another manner then single helices. <br>
We also measured the lengths of the origami structures on TEM images. Histograms of the length distributions display a gaussian shape (figure 6). For increasing concentrations of spermine, the length decreases steadily (figure 6a, for raw data see this file: <html><a href="http://openwetware.org/images/b/b6/TEM_length_measurements_raw_data.xlsx">TEM_length_measurements_raw_data.xlsx</a></html>. Surprisingly, for rising concentrations of ethidium bromide, the length decreases as well, although addition of ethidium bromide is usually known to increase the length of a simple double stranded DNA (figure 6b). It seems that DNA origami structures behave different from single DNA helices in this regard. <br>


[[Image:Control length histo gaussian.png |390px|center|thumb|Fig. 6 Length distribution of theU negative control, with a gaussian fit, histogram based on 256 particles]]<br>
[[Image:Control length histo gaussian.png |390px|center|thumb|Fig. 6 Length distribution of theU negative control, with a gaussian fit, histogram based on 256 particles]]<br>
Line 119: Line 119:
<h2>FRET Bulk Measurements</h2>
<h2>FRET Bulk Measurements</h2>
For first tests, a simple [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Structure_page#MH_255_and_MH_256 18 bp DNA double helix] with Atto 550 ddCTP at the one end and Atto 647N ddUTP at the other end was examined.
For first tests, a simple [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Structure_page#MH_255_and_MH_256 18 bp DNA double helix] with Atto 550 ddCTP at the one end and Atto 647N ddUTP at the other end was examined.
The idea to perform bulk measurements based on FRET using a photospectrometer and a real time PCR was unsuccessful.  
The idea to perform bulk measurements based on FRET using a photospectrometer and a real time PCR was not successful.  
The photospectrometer is not sensitive enough to handle Atto dyes at concentrations below 10 nM (peaks were not visible at all).  
The photospectrometer turned out not to be sensitive enough to handle Atto dyes at concentrations below 10 nM (peaks were not visible at all).  
The real time PCR, which is more sensitive, still did not deliver trustworthy data when using 50 µl samples with 10 nM Atto dyes. It could be shown that the reproducibility of the real time PCR setup was poor with deviations of up to 40 % between identical samples (figure 7). To assure the identity of the samples a 100 µl stock was divided into two 50 µl samples. Based on these results no experiments with theU structure were performed at all with this device as the concentration of theU structure is lower than the concentration of the here test structure.  
The real time PCR, which is more sensitive, still did not deliver trustworthy data when using 50 µl samples with 10 nM Atto dyes. It could be shown that the reproducibility of the real time PCR setup was poor with deviations of up to 40 % between identical samples (figure 7, to assure the identity of the samples, a 100 µl stock was divided into two 50 µl samples). Based on these results no experiments with theU structure were performed at all with this device as the concentration of theU structure is lower than the concentration of the here test structure.  


<table cellspacing="0" cellpadding="2">
<table cellspacing="0" cellpadding="2">
Line 128: Line 128:




To handle the issue with the small concentrations further experiments were done with a fluorescence microscope.
To handle the issue of small concentrations, further experiments were performed with a fluorescence microscope.


<h2>Single Molecule Measurements at the Fluorescence Microscope</h2>
<h2>Single Molecule Measurements at the Fluorescence Microscope</h2>
Line 142: Line 142:


<table cellspacing="0" cellpadding="2">
<table cellspacing="0" cellpadding="2">
<tr bgcolor="#f5f5f5"><td valign="top">The analysis program is a [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Methods#Evaluation_of_Data matlab script] which searches for spots in the red and the green movie and plots the intensities over time to identify bleaching events. Only those plots where the acceptor bleaches first and the donor bleaches afterwards are useful to calculate the FRET-efficiency (see figure 9). <br>The graph shows the intensities of the donor and the acceptor and in addition the intensity of the FRET-events. As one can see the intensity of the donor rises as soon as the acceptor bleaches. After some while the donor bleaches too. From that the FRET-efficiency can be calculated.<br>
<tr bgcolor="#f5f5f5"><td valign="top">The analysis program is a [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Methods#Evaluation_of_Data matlab script] which searches for spots in the red and the green movie and plots the intensities over time to identify bleaching events. Only those plots where the acceptor bleaches first and the donor bleaches afterwards are useful to calculate the FRET-efficiency (see figure 9). <br>The graph shows the intensities of the donor and the acceptor and, in addition, the intensity of the FRET-events. As one can see, the intensity of the donor rises as soon as the acceptor bleaches. After some while the donor bleaches too. From that, the FRET-efficiency can be calculated.<br>


First we measured the FRET-efficiencies for the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Structure_page#BM12_to_BM14:_Fluorophore-labeled_Structures_for_FRET_Measurements.2C_including_Adapters_for_Immobilisation BM14 structure] without any intercalator or groove binder as a control and afterward we measured the same structure with 4.8µM spermine (corresponding to one molecule every 7bp). The FRET-efficiencies were plotted in figure 9.</td><td>[[Image:Schöner_FRET_Verlauf.PNG|400px|thumb|Fig. 9 Intensities of donor and acceptor]]</td></tr>
First we measured the FRET-efficiencies for the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Structure_page#BM12_to_BM14:_Fluorophore-labeled_Structures_for_FRET_Measurements.2C_including_Adapters_for_Immobilisation BM14 structure] without any intercalator or groove binder as a control and afterward we measured the same structure with 4.8µM spermine (corresponding to one molecule every 7bp). The FRET-efficiencies were plotted in figure 10.</td><td>[[Image:Schöner_FRET_Verlauf.PNG|400px|thumb|Fig. 9 Intensities of donor and acceptor]]</td></tr>
</table>
</table>


Line 152: Line 152:




It is obvious that we actually measured FRET, though the low yield of FRET-events that were found by the matlab script does not allow to draw any conclusions because of the low statistics. The wide spread of FRET efficiencies is probably caused by the base twists observed in the TEM measurements. Here further optimization needs to be done. Yet the fact that there actually were FRET-events makes it worth to keep on elaborating these measurements.
It is obvious that we actually measured FRET, though the low yield of FRET-events that were found by the matlab script does not allow to draw any conclusions because of low-number statistics. The wide spread of FRET efficiencies is probably caused by the base twists observed in the TEM measurements. Here further optimization is neccessary. Yet the fact that there actually were FRET-events makes it worth to keep on elaborating these measurements.


<h3>Fluorescence Tracking</h3>
<h3>Fluorescence Tracking</h3>


Besides FRET-measurements we also applied another approach to investigate the deformation of the structure where we determined the distance between the fluorophores and thereby get the distance of the two arms by directly comparing two images. At first we excited the Atto 550 dye and observed at its characteristic wavelength, subseqeuntly Atto 647N was excited and observed. For the analysis with the homemade matlab script at first we had to [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/2011/10/19#Camera_calibration calibrate the cameras].
Apart from FRET-measurements, we also applied another approach to investigate the deformation of the structure where we determined the distance between the fluorophores and thereby get the distance of the two arms by directly comparing two images. At first, we excited the Atto 550 dye and observed at its characteristic wavelength, subseqeuntly Atto 647N was excited and observed. For the analysis with the homemade matlab script, at first we had to [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/2011/10/19#Camera_calibration calibrate the cameras].
Then the matlab script searched for spots in the green and the red picture and fitted them with a gaussian. The peaks from the green picture then are transfered into the red picture. When there is a matching red spot for the green spot the distance between them is calculated.
Then the matlab script searched for spots in the green and the red picture and fitted them with a gaussian. The peaks from the green picture are subsequently overlaid with the red picture. When there is a matching red spot for the green spot the distance between them is calculated.
We did those measurements for a control and for two different concentrations of spermine.
We did those measurements for a control and for two different concentrations of spermine.
Quantitative evidence is a bit tricky because of the calibration and the fact that one pixel of the pictures equals 101.03nm. Nevertheless we decided to take pictures in epifluorescence mode of a negative control without DNA binders and with two different spermine concentrations (one spermine every 7 bases and one spermine every 21 bases). Every picture was illuminated for 1 sec with the green laser for the green channel and then with the red laser for the red channel for the same time. The graph below (figure 11) shows the histograms of the distribution of the distance between the maxima of the fitted gaussians in the green and red channel. <br>
Quantitative evidence is a bit tricky because of the calibration and the fact that one pixel of the pictures equals 101.03nm. Nevertheless, we decided to take pictures in epifluorescence mode of a negative control without DNA binders and with two different spermine concentrations (one spermine every 7 bases and one spermine every 21 bases). Every picture was illuminated for 1 sec with the green laser for the green channel and then with the red laser for the red channel for the same time. The graph below (figure 11) shows the histograms of the distribution of the distance between the maxima of the fitted gaussians in the green and red channel. <br>


[[Image:Gauss distance hist.png|500px|center|thumb|Fig. 11 Distance distributions between the fluorescent dyes with varying spermine concentrations]]<br>
[[Image:Gauss distance hist.png|500px|center|thumb|Fig. 11 Distance distributions between the fluorescent dyes with varying spermine concentrations]]<br>


The distributions look nearly the same for every concentration except for the control. This is due to the small number of points that were measured for these traces. Furthermore the values for each trace seam not to be distributed in a gaussian manner. This maybe underlies the electrostatic repulsion of the arms when they are in close vicinity. Also the distribution reaches up to 120 nm. This is not realistic. Possible reasons for this artifacts could be misalignments of the pictures and not accurate enough determination of the spots since we wanted to measure spatial separations in the regime of 5 nm which corresponds to a 20th of one single pixel on the detector. Also acquisition of uncorrelated spots which belong to different structures might be a problem. So one has to refine the setup and acquire more values for better statistics to get trustable values of a mean distance of the arms.
The distributions look nearly the same for every concentration, except for the control. This is due to the small number of points that were measured for these traces. Furthermore, the values for each trace seam not to be distributed in a gaussian manner. This maybe underlies the electrostatic repulsion of the arms when they are in close vicinity. Also the distribution reaches up to 120 nm. This is not realistic. Possible reasons for this artifacts could be misalignments of the pictures and not accurate enough determination of the spots since we wanted to measure spatial separations in the regime of 5 nm which corresponds to a 20th of one single pixel on the detector. Also acquisition of uncorrelated spots which belong to different structures might be a problem. So one has to refine the setup and acquire more values for better statistics to get trustable values of a mean distance of the arms.


<h1>Discussion</h1>
<h1>Discussion</h1>

Latest revision as of 23:59, 2 November 2011

<html> 

  <style>
  1. column-one { display:none; width:0px;}

.container{background-color: #f5f5f5; margin-top:50px} .OWWNBcpCurrentDateFilled {display: none;}

  1. content { width: 0px; margin: 0 auto auto 0; padding: 1em 1em 1em 1em; align: center;}
  1. column-content {width: 0px; float: left; margin: 0 0 0 0;padding: 0;}

.firstHeading {display:none; width:0px;}

  1. globalWrapper{ width:1280px;}

div.tright { border-bottom-width: 0em; border-left-width: 0px; border-right-width: 0px; border-top-width: 0em; clear: right; float: right; 

}

div.tleft { border-bottom-width: 0em; border-left-width: 0px; border-right-width: em; border-top-width: 0em; clear: left; float: left; margin-right: 0.5em; 

}

div.tnone { border-bottom-width: 0em; border-left-width: 0px; border-right-width: em; border-top-width: 0em; }

  1. column-one {display:none; width:0px;background-color: #ffffff;}
  1. content{ margin: 0 0 0 0; padding: 1em 1em 1em 1em; position: center; width: 800px;background-color: #f5f5f5; }

.container{ width: 800px; margin: auto; background-color: #ffffff; text-align:justify; font-family: helvetica, arial, sans-serif; color:#555555; margin-top:25px; }

  1. bodyContent{ width: 1267px; align: center; background-color: #ffffff;}
  1. column-content{width: 1280px;background-color: #f5f5f5;}

.firstHeading { display:none;width:0px;background-color: #ffffff;}

  1. header{position: center; width: 800px;background-color: #ffffff;}
  1. footer{position: center;}

<style type="text/css"> body { 

   font-family: helvetica, arial, sans-serif;
   color: black;

background:#f5f5f5; 

 }

ul#topnav {

margin: 0 0 0 0px ; padding: 0 0 0 0px; float: left; width: 800px; list-style: none; position: relative; 

       font-family: helvetica, arial, sans-serif;

font-size: 12px; background: #f5f5f5; 

       height: 5px;

} ul#topnav li { float: left; margin: 0; padding: 0; border-right: 1px solid #ffffff; /*--Divider for each parent level links--*/ } ul#topnav li a { padding: 11px 15px; display: block; color: #333333; text-decoration: none; } ul#topnav li:hover { background: #f5f5f5;} /*--Notice the hover color is on the list item itself, not on the link. This is so it can stay highlighted even when hovering over the subnav--*/

ul#topnav li span { float: left; padding: 15px 0; position: absolute; left: 0; top:30px; display: inline; /*--Hide by default--*/ width: 800px; background: #cccccc; color: #333333; /*--Bottom right rounded corner--*/ -moz-border-radius-bottomright: 5px; -khtml-border-radius-bottomright: 5px; -webkit-border-bottom-right-radius: 5px; /*--Bottom left rounded corner--*/ -moz-border-radius-bottomleft: 5px; -khtml-border-radius-bottomleft: 5px; -webkit-border-bottom-left-radius: 5px; } ul#topnav li:hover span { display: block; } /*--Show subnav on hover--*/ ul#topnav li span a { display: inline;} /*--Since we declared a link style on the parent list link, we will correct it back to its original state--*/ ul#topnav li span a:hover {text-decoration: underline;}


  1. abstractandlinks{

display:block; width:800px; background-color:#f5f5f5; text-align:justify;font-family: helvetica, arial, sans-serif; color:#555555; margin-top:0px; }

  1. innerBox

{ display: block; padding: 5px; }

  1. abstract{

font-family: helvetica, arial, sans-serif; width: 490px; margin-top: 20px; padding:0px 5px; float: left;background-color:#f5f5f5; ; }

  1. links{

font-family: helvetica, arial, sans-serif; width: 290px; margin-top: 20px; padding:0px 5px; float: left; background-color:#f5f5f5;; } .clear { clear:both; }

  1. tour{

font-family: helvetica, arial, sans-serif; width: 790px; padding:0px 5px; margin-top: 1em, margin-bottom: 1em; float: left;background:#f5f5f5; font-size: 2em; }

  1. struktur{

font-family: helvetica, arial, sans-serif; width: 790px; padding:0px 5px; float: left;background:#f5f5f5; }

  1. toctitle {align: left; text-align: left; }
  2. table.toc {background-color:"#5f5f5f";align: left; text-align: left;}
  3. toc #toctitle, .toc #toctitle, #toc .toctitle, .toc .toctitle {

text-align: left; 

}
  1. toc, .toc, .mw-warning {

background-color: #f5f5f5; border-bottom-color: rgb(170, 170, 170); border-bottom-style: solid; border-bottom-width: 0px; border-left-color: rgb(170, 170, 170); border-left-style: solid; border-left-width: 0px; border-right-color: rgb(170, 170, 170); border-right-style: solid; border-right-width: 0px; border-top-color: rgb(170, 170, 170); border-top-style: solid; border-top-width: 0px; font-size: 105%; padding-bottom: 5px; padding-left: 5px; padding-right: 5px; padding-top: 5px; align: left; text-align: left; display: left 

}


</style>

<head>

<script type="text/javascript">

$(document).ready(function() {

$("ul#topnav li").hover(function() { //Hover over event on list item $(this).css({ 'background' : '#cccccc'}); //Add background color and image on hovered list item $(this).find("span").show(); //Show the subnav

} ,

$("ul#topnav li").oneclick(function() { //Hover over event on list item $(this).css({ 'background' : '#cccccc'; 'display' : 'inline'}); //Add background color and image on hovered list item $(this).find("span").show(); //Show the subnav

} ,

function() { //on hover out...


$(this).css({ 'background' : 'none'}); //Ditch the background

//$(this).find("span").hide(); //Hide the subnav });

}); </script>

<title> 

       TUM NanU - Home

</title> </head>

<body background-color="#F0F0F0"> <!-- Header--> <div class="container"> 

   <div id="header"> <img src="http://openwetware.org/images/5/5c/Header2.png" /> </div>

<!--Menue--> <ul id="topnav"> 

   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Home">Home</a></li>
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Project">Project</a>        
        
   </li>

   <li style="background-color: #cccccc"><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results"><b>Results & Discussion</b></a>
       <span>
        
          
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#Folding_.26_Purification">Folding & Purification</a>| 
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#TEM_Image_Analysis">TEM Image Analysis</a>| 
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#Fluorescence_Measurements">Fluorescence Measurements</a>|
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#Discussion">Discussion</a>|
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#Outlook">Outlook</a>
       </span>
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Methods">Methods</a>
     
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Team">Team</a></li>
 <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA">Labbook</a><!--Subnav Starts Here-->
       
   </li> 
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Extras">Extras</a>
       
  </li>

</ul> <p> <br> </p> <p> <br> </p> <p> <br> </p>

</body> </html>

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Biomod/2011/TUM/TNT/Results&oldid=564062"