Amino allyl - UTP Labelling of RNA

Peter Hauser (last update 17.12.2002)

according to


Primer annealing:

  • aRNA (800 ng) was primed with random nonamers (1µl), nuclease free water was added to get a total volume of 11µl
  • Incubate the annealing mix at 70°C for 5 minutes
  • Cool the mix at room temperature for 10 minutes
  • Spin down in microcentrifuge
  • Add 4 µl 5x CyScript buffer, 2µl 0.1 M DTT, 1 µl Nucleotide mix, 1 µl aminoallyl-dUTP and 1µl CyScript reverse transcriptase
  • Incubate reaction mix at 42°C for 90 minutes

RNA degradation:

  • Add 2µl 2.5 M NaOH to the reaction mix
  • Mix by vortexing and spin down
  • Incubate at 37°C for 15 minutes
  • Add 10µl 2M HEPES to the reaction
  • Mix by vortexing and spin down

Purification of aminoallyl cDNA:

  • Add 3 µl 3M sodium acetate to each sample
  • Add 75µl absolute athanol
  • Incubate at -70°C for 60 minutes or overnight
  • Centrifuge with 12.000 g for 30 minutes at 4°C
  • Remove supernatant and add 1ml 70% ethanol
  • Centrifuge with 12.000 g for 15 minutes at 4°C
  • Air dry pellet and resuspend in 15µl nuclease free water

Labelling reaction:

  • Resuspend CyDye aliquot in 15µl 0.1 M sodium bicarbonate pH9.0
  • Add CyDye to the purified cDNA and mix by stirring
  • Incubate for 60 minutes in the dark
  • Add 15µl 4 M hydroxlamine to the reaction

Purification of labelled cDNA using QIAquick PCR purification kit:

  • Add 225µl buffer PB to the reaction
  • Apply the mixture on to QIAquick columns
  • Spin for 60 seconds with 10.000g at RT
  • Discard flow through and add 650µl wash buffer PE to the column
  • Centrifuge 60 seconds with 10.00g at RT
  • Discard flow through and add 500µl wash buffer PE to the column
  • Centrifuge 60 seconds with 10.00g at RT
  • Transfer column to a new tube and add 50 µl nuclease free water
  • Incubate 1 minute at room temperature, than centrifuge 60 seconds with 10.000 g at RT

Purified labelled cDNA is ready for use.



CyScribe cDNA Post Labelling Kit Amersham, Buckinghamshire, UK #RPN5660
QIAqucik PCR Purification Kit Qiagen, Hilden, Germany #28104
Ethanol Merck, Darmstadt, Germany # 1.00983.1011
Nuclease free Water GibcoBRL Gaithersburg, MD #10977-015
3M Sodium Actetat Sigma, Steinheim, Germany #S-7899
Sodium Bicarbonate GibcoBRL Gaithersburg, MD #11810-025
HEPES USB, Cleveland, Ohio #16928
Sodiumhydroxid Merck, Darmstadt, Germany #6482.100