Biomod/2011/TUM/TNT/LabbookA/2011/08/24: Difference between revisions

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== labeling oligos with fluorophores ==
== labeling oligos with fluorophores ==
* labeling using terminal transferase according to protocol in BioNanoWiki, but 2x the usual volumes (final volume: 80µl)
labeling using terminal transferase according to [http://openwetware.org/wiki/Labeling_oligonucleotides_with_fluorophores standard protocol].<br>
* all reactions were accomplished twice, but for the_U 218 and the_U 225 only one reaction was successful
all reactions were accomplished twice, but for the_U 218 and the_U 225 only one reaction was successful <br>
* labeled oligos:  
labeled oligos:  
<br>
<br>
* the_U 175 with Atto550_ddCTP      helix 4
* the_U 175 with Atto550_ddCTP      helix 4
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* the_U 204 with Atto647N_ddUTP      helix 20
* the_U 204 with Atto647N_ddUTP      helix 20
<br>
<br>
*for purification, batches of identical probes were combined
for purification, batches of identical probes were combined <br>
*labeled oligos were resuspended in 100µl 0.5xTBE 11mM MgCl2 (50µl for the_U 218 and the_U 255)
labeled oligos were resuspended in 100µl 0.5xTBE 11mM MgCl2 (50µl for the_U 218 and the_U 255) <br>
* further see [http://openwetware.org/wiki/Tnt_-_Labbook/2011/08/25#Determined_Concentrations_of_Labeled_Oligos concentration measurement]
<br>
<br>
*prepared solutions for determining absorption coefficients of unlabeled oligonucleotides according to [http://openwetware.org/wiki/Concentration_measurement_of_labeled_oligonucleotides standard protocol]
prepared solutions for determining absorption coefficients of unlabeled oligonucleotides according to [http://openwetware.org/wiki/Concentration_measurement_of_labeled_oligonucleotides standard protocol]




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labeling oligos with fluorophores

labeling using terminal transferase according to standard protocol.
all reactions were accomplished twice, but for the_U 218 and the_U 225 only one reaction was successful
labeled oligos:

  • the_U 175 with Atto550_ddCTP helix 4
  • the_U 218 with Atto647N_ddUTP helix 23


  • the_U 197 with Atto550_ddCTP helix 8
  • the_U 225 with Atto647N_ddUTP helix 29


  • the_U 183 with Atto550_ddCTP helix 5
  • the_U 204 with Atto647N_ddUTP helix 20


for purification, batches of identical probes were combined
labeled oligos were resuspended in 100µl 0.5xTBE 11mM MgCl2 (50µl for the_U 218 and the_U 255)

prepared solutions for determining absorption coefficients of unlabeled oligonucleotides according to standard protocol

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