Made a fresh, double batch (50uL rxn instead of 25uL) of cDNA according to Promega MMLV protocol using oligo dT primers. cDNA was put into a plate for faster qPCR loading. cDNA calcs and plate layout are here. Briefly, RNA and oligo dTs were combined, brought up to 37uL, heated @ 70C for 5mins and immediately placed on ice. RT master mix was made (RT master mix calcs are here), 13uL was distributed to each well. Samples were incubated @ 42C for 1hr and then 95C for 5mins.
Apparently, these samples had not been spec’d after DNase treatment.
Samples range in quality (260/280) from not great to perfect. Will perform calcs to make cDNA.
Set up reverse transcription rxns using 174ng of each DNased RNA (sample 07:12-04 was limiting; only 6.1uL available), using Promega oligo dT primers and M-MLV Reverse Transcriptase according to Promega protocol. RNA/oligo dT primer workup is 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, incubated 42C for 1hr., 95C for 3mins and then the samples were given to Lisa in the Friedman Lab.
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.
Performed RT using Promega M-MLV RT according to M-MLV protocol and used 0.5ug oligo dT per ug of RNA on all BB and DH site samples that were negative for gDNA (see qPCR results 20090512). Calculations and work up are here. Samples were set up in a plate to facilitate sample loading in subsequent qPCRs.
Set up the MMLV RT rxns with random primers using ~833ng DNAsed RNA (prepared yesterday) according to the Promega MMLV Product Insert. This procedure is slightly different than what is in our lab protocol for RT rxns. Here is the workup for the rxns. cDNA was stored @ -20C in the “Vibrio” box.