Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.
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Hi, I'm new to molecular biology and one of my first tasks is to generate a cDNA clone of a particular human gene. I started off by isolating total RNA from whole blood by the Trizol method. Ran the RNA on a denaturing gel and the RNA is beautiful....can distinctly see the bands for rRNA and there is practically no smearing or genomic contam whatsoever. Gave DNase treatment followed by a cleanup (Qiagen). The final concentration of RNA is 614ng/ul. I take 2ul of this and attempt to make cDNA by the Thermo Scientific Verso kit and get a big fat nothing! All my control PCRs (actin and rRNA) are blank and for the life of me I can't figure out why. Does anyone have any bright ideas? Am I using too little starting matterial (kit specifies 1ug or less)? Is my RNA getting degraded during the DNase treatment?
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