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

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<h1>Labeling</h1>
<h1>Labeling oligonucleotides with fluorophores</h1>
For fluorecence analysis and FRET measurements, the dyes need to be attached to the origamis in a defined manner. Therefore, dyes were bound to the 3' ends of selected staples. Dye-labeled oligonucleotides are commercially available, but quite expensive. On the one hand, we need only small amounts of labeled staples, on the other hand a variety of ten different oligonucleotides was used for different experiments. Under these circumstances, it was preferable to buy the both dyes each bound to one ddNTP and then couple them to the oligonucleotides of interest. This could be achieved using terminal transferase, which is also [https://www.roche-applied-science.com/servlet/RCProductDisplay?storeId=10202&catalogId=10202&langId=-1&countryId=us&forCountryId=us&productId=3.6.14.3.7.2 commercially available]. This enzyme binds (modified) dNTPs or ddNTPS to free 3' ends. <br>
For fluorecence analysis and FRET measurements, the dyes need to be attached to the origamis in a defined manner. Therefore, dyes were bound to the 3' ends of selected staples. Dye-labeled oligonucleotides are commercially available, but quite expensive. On the one hand, we need only small amounts of labeled staples, on the other hand a variety of ten different oligonucleotides was used for different experiments. Under these circumstances, it was preferable to buy the both dyes each bound to one ddNTP and then couple them to the oligonucleotides of interest. This could be achieved using terminal transferase, which is also [https://www.roche-applied-science.com/servlet/RCProductDisplay?storeId=10202&catalogId=10202&langId=-1&countryId=us&forCountryId=us&productId=3.6.14.3.7.2 commercially available]. This enzyme binds (modified) dNTPs or ddNTPS to free 3' ends. <br>
After [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Labeling_oligonucleotides_with_fluorophores labeling staples], the yield was determined by measuring absorption at 260nm. In this [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Concentration_measurement_of_labeled_oligonucleotides procedure], at first an absorption coefficient for the unlabeled oligonucleotide is determined, since these coefficients may display huge deviations from those obtained from online tools or suppliers. The coefficient of the one ddNTP with attached dye was determined separately. By adding these values, ε<sub>260</sub>(labeled oligonucleotide) could be estimated and used for concentration measurement of the labeled staples.  
After [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Labeling_oligonucleotides_with_fluorophores labeling staples], the yield was determined by measuring absorption at 260nm. In this [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Concentration_measurement_of_labeled_oligonucleotides procedure], at first an absorption coefficient for the unlabeled oligonucleotide is determined, since these coefficients may display huge deviations from those obtained from online tools or suppliers. The coefficient of the one ddNTP with attached dye was determined separately. By adding these values, ε<sub>260</sub>(labeled oligonucleotide) could be estimated and used for concentration measurement of the labeled staples.  

Revision as of 14:57, 27 October 2011

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