Prepared cDNA using 1ug of RNA from each of the 3 pools (CA, MA, MAX) and processed according to Promega’s M-MLV protocol, using oligo dT primers. Calcs and master mix set up are here. Briefly, RNA was combined with oligo dT primers, denatured @ 70C for 5mins, immediately placed on ice for 2mins, mixed with RT master mix, incubated 1hr @ 42C, 3mins @ 95C, and then stored @ -20C.
Tag Archives: M-MLV
Reverse Transcription – BB & DH DNased RNA (from 20090514)
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.
UPDATE: cDNA plate was discarded 20120320 by SJW.
Reverse Transcription – Abalone 07:12 DNased RNA (from 20090623)
Spec – DNased Abalone 07:12 RNA
Apparently, these samples had not been spec’d after DNase treatment.
Results:
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.
Reverse Transcription – Sepia DNased RNA
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.
Results:
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.
Reverse Transcription – Tim’s adult gigas challenge DNased RNA (from 20091008)
Performed RT rxns. Setup is here. cDNA was subsequently diluted 1:4 (added 75uL H2O to each well) to properly mirror Tim’s previous qPCRs.
UPDATE: cDNA plate was discarded 20120320 by SJW.
RT Rxns – H.crach DNased RNA (from 20090623)
DNased RNA was used for cDNA rxns. Workup of the amount of RNA used in each rxn (472ng/uL) is here (Google Spreadsheet), along with previous cDNA batch numbers. Actual RT master mix set up is here. Samples were given to Lisa for qPCR analysis.
Reverse Transcription – MV hemocyte DNased RNA from 20090612
Made cDNA using Promega M-MLV RT and oligo dT primers. RT rxn set up is here. Samples were stored @ 4C until ready to use.
Reverse Transcription – Mac’s gigas DNased RNA from 20090512
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.
UPDATE: cDNA plate was discarded 20120320 by SJW.
Reverse Transcription – cDNA from DNased Abalone RNA from 20090420
Prepared cDNA using an equal amount of RNA from all samples (442.6ng). This amount was based on using the maximum allowable volume of RNA for the RT rxn AND the sample with the lowest [RNA] (08:3-20; 24.59ng/uL). cDNA was prepared according to the Promega MMLV RT recommendations. Here is the work up for the cDNA rxns. cDNA was stored @ -20C.
Reverse Transcription – V.tubiashii DNAsed RNA (from yesterday)
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.
