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

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<h3>Folding Batch</h3>
<h3>Folding Batch</h3>
The folding batch contains [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Glossary#F FOB20], 20nM scaffold and 200nM of each staple. Dye-labeled oligonucleotides are added extra at this step.  
The folding batch contains [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Glossary#FOB FOB20], 20nM scaffold and 200nM of each staple. Dye-labeled oligonucleotides are added extra at this step.  


<h3>Thermal Ramp</h3>
<h3>Thermal Ramp</h3>
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The usual procedure for purification of folded origamis uses agarose gel electrophoresis. Thereby the main product is separated from single staples and some misfolded structures, e.g. stably linked dimers. While electrophoresis is a helpful tool for analysis of the folding products, for preparative means it has the disadvantages of low yields (estimated to be roughly 10% of the loaded structure) and, more importantly, the samples are contaminated with ethidium bromide. We investigated our origami with gel electrophoresis and found only one weak band in addition to the correctly folded structure. With such few misfolding events, the aim of purification was only to remove unused staples. This could be reached by using size exclusion filters with 100kDa molecular weight cutoff. This new procedure saves time (ca. 1/2 hour as compared to several hours), is less expensive than resolubilization out of agarose gels, loss of product is significantly reduced, and the origamis are not contaminated with intercalators. <br>
The usual procedure for purification of folded origamis uses agarose gel electrophoresis. Thereby the main product is separated from single staples and some misfolded structures, e.g. stably linked dimers. While electrophoresis is a helpful tool for analysis of the folding products, for preparative means it has the disadvantages of low yields (estimated to be roughly 10% of the loaded structure) and, more importantly, the samples are contaminated with ethidium bromide. We investigated our origami with gel electrophoresis and found only one weak band in addition to the correctly folded structure. With such few misfolding events, the aim of purification was only to remove unused staples. This could be reached by using size exclusion filters with 100kDa molecular weight cutoff. This new procedure saves time (ca. 1/2 hour as compared to several hours), is less expensive than resolubilization out of agarose gels, loss of product is significantly reduced, and the origamis are not contaminated with intercalators. <br>
For detailed information on the procedure, please read the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Purification_of_origamis_by_size_exclusion_filtration#Purification_of_origamis_by_size_exclusion_filtration instruction] in our labbook. Results of the analysis of filtered structures with agarose gel electrophoresis can be found [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results/Folding_Purification#Purification_with_size_excluscion_filters here].  
For detailed information on the procedure, please read the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Purification_of_origamis_by_size_exclusion_filtration#Purification_of_origamis_by_size_exclusion_filtration instruction] in our labbook. Results of the analysis of filtered structures with agarose gel electrophoresis can be found [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#Folding_.26_Purification here].  


<h1>Labeling</h1>
<h1>Labeling</h1>
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For measuring the distance the MATLAB software was modified. After picking the spots in each picture they were aligned and for each spot the intensity was fitted with gaussian curve. Afterwards the distance  was calculated between the peaks of two matching spots.  
For measuring the distance the MATLAB software was modified. After picking the spots in each picture they were aligned and for each spot the intensity was fitted with gaussian curve. Afterwards the distance  was calculated between the peaks of two matching spots.  


[[Image:Tirfauswertung.jpg|600px|center|thumb|Fig. 2 evaluation of data received from ALEX for distance calculation]]
[[Image:Tirfauswertung.jpg|600px|center|thumb|Fig. 3: evaluation of data received from ALEX for distance calculation.]]




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