RNA Newbie questions - (Jul/02/2001 )
I would like to sequence a mRNA transcript from a gene of interest. The transcript should be about 2KB in length.Never having done this before, I could use a little guidance. It looking like Gibco all the way around (unless thereare better suggestions): Trizol for the total RNA isolation from mouse spleen, MessageMaker kit to get the mRNA,and Superscript II to make the cDNA.
My question is to how I would sequence the total mRNA transcript? I am not interested in quantifying it, so I willnot need to do a Northern or RT-PCR at this stage. Should I use a kit like the TOPO TA and insert the mRNAinto a vectors and then probe with sequence from the gene of interest? Or could you use RACE to amplify theentire transcript? A little help/suggestions?
the preparation agents are used by me, too and they are working fine.so if you make cDNA from your transcript, why don`t you sequencing this cDNA, formerly cloned in a TA-cloning vector?