DNase Treatment - Sepia RNA (from 20091204)
Samples were DNase treated with Ambion’s Turbo DNA-free kit, following the rigorous protocol. Used 6uL of each sample, brought up to 50uL with H2O, added 5uL of 10x buffer and 1.5uL of DNase. Incubated 37C for 30mins, added an additional 1uL of DNase and incubated @ 37C for 30mins. Added 0.2 volumes of DNase Inactivation reagent and incubated at RT for 2mins with regular mixing. Spec’d RNA.
Set up reverse transcription rxns using 200ng of each DNased RNA, using Promega oligo dT primers and M-MLV Reverse Transcriptase according to Promega protocol. RNA/Oligo dT primer workup here. Primer and DNAsed RNA were mixed and brought up to 18.25uL with H2O. Samples were heated @ 70C for 5mins and then placed immediately on ice. The RT master mix set up can be found here. 6.75uL of the RT master mix was added to each tube, mixed, spot spun and then incubated @ 42C for 1hr, heat inactivated @ 95C for 3mins and stored @ 4C.