Biomod/2011/TUM/TNT/LabbookA/Labeling oligonucleotides with fluorophores

From OpenWetWare

Jump to: navigation, search

TUM NanU - Home




Contents

Labeling oligonucleotides with fluorophores

Intention

We use always the same two fluorophors, Atto 550 and Atto 647N, for our FRET measurements. However, we need them attached to different staples for our FRET measurements. Therefore, we bought fluorophore labeled nucleotides, Atto 550 ddCTP and Atto 647N ddUTP. Using terminal tranferase, these labeled nucleotides can ba added at the 3' end of a freely chosen oligonucleotide.

Protocol

mix

  • 16µl 5x TdT reaction buffer (supplied with terminal tranferase)
  • 16µl 25mM CoCl2
  • 4µl 100µM oligonucleotide that should be labeled
  • 2nmol fluorophor labeled nucleotide, i.e. 2µl 1mM Atto 550 ddCTP or 4µl 0.5mM Atto 647N ddUTP
  • 2µl terminal transferase
  • add ddH2O to a final volume of 80µl


incubate at 37°C for 40 minutes
add 8µl 0.2mM EDTA (pH8)and incubate at 70°C for 10 minutes in order to stop reaction

For purification, the oligo is precipitated in ethanol and then washed several times.

  • add 25µl 3M NaOAc and 275µl 99+% EtOH
  • incubate on dry ice for 30 min
  • centrifuge for 30 min at 4°C at 16100 rcf
  • carefully remove the supernatant and add 400µl 75% EtOH in water
  • vortex thoroughly
  • centrifuge for 30 min at 4°C at 16100 rcf
  • carefully remove the supernatant and add 400 µl 75% EtOH in water
  • vortex thoroughly
  • centrifuge for 30 min at 4°C at 16100 rcf
  • carefully remove the supernatant and dissolve the pellet in 50µl 0.5x TBE 11mM MgCl2
  • incubate over night at 4°C until the oligonucleotide has completely dissolved
  • hide sample frim light to prevent photobleaching