I am facing a problem with quantification of real-time PCR data using rotor gene-Q machine. I am using this technology for the first time. Please suggest me what are the preliminary steps required to initiate the work. I've isolated RNA from plant at three different stage of heat treatment and want to compare the results of all. All the kit were bought from Qiagen. In my experiment tubulin was used as standard but when I go for auto detect threshold level the program say that at least two standards are required to find auto detect threshold level. I've complemented my analysis with all the treatment using 24 targeted gene with one standard. Could you please suggest me how should I process my data for final analysis to publish in scientific journal.
If you are going for cloning of isolated DNA then its better to remove RNA from DNA sample but if you working with molecular marker analysis just dissolve the pallet of isolated DNA in TE buffer already contain RNase (15ul of 10mg/ml Rnase in 100ml of TE bufer). You will get good results. Tell me on what you are working.
i wonder if i would like to get pure gDNA without RNA contamination, in which step should i add RNase?
The protocol that i'm using: --> lysozyme treatment overnight --> add SDS/proteinase k + RNase and incubate at 370C for 30 min --> incubate at 560C for 3 hours --> phenol/chloroform extraction x 3 --> chloroform extraction --> sodium acetate + isopropanol
i'm still getting some rRNA contamination when view under gel. Wonder what's wrong in my extraction steps. Anybody could give any advice? Thanks.
I isolated plant leaf RNA using RNeasy kit of qiagen. Kit itself assure DNA contamination free RNA but some minut quantity I recorded on my agarose gel. I've not done on-column DNase treatment but now I want to treat my samples with DNase. Can I use NEB DNase for this RNA. second RNA cleanup procedure is required after this treatment or not? How much RNA I should treat with DNase?