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.
Tag Archives: reverse transcription
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.
cDNA – Abalone RNA from 20090331 & 20090402
cDNA was made from the above RNA samples using the Qiagen Quantitect RT Kit. The samples were laid out in a PCR plate. 274.2ng of RNA was used in the rxn for each sample, based on the lowest concentration RNA sample (08:3-20) to equalize all the samples. The Genomic Wipeout step of the kit requires 2uL of Genomic Wipeout enzyme/buffer to be added to 12uL of an RNA sample, so the calculations were done and can be found here. A check mark on the calculation sheet indicates that the water and then the RNA was added to the appropriate wells. Those with two check marks were used for a “No RT” rxn and thus, have duplicate wells (see plate layout).
The plate was mixed, spot spun, uncubated at 42C for 2mins and immediately placed on ice.
The RT and No RT master mixes were set up on ice and then added to the respective wells (see sheet here).
UPDATE: cDNA plate was discarded 20120320 by SJW.
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.
