Peter Hauser (last update 17.12.2002)

according to http://cmgm.stanford.edu/pbrown/protocols/index.html

Procedures

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.

Materials: