Hi!!

I am trying to do radioactivity based invitro transcription assay using oligo dc tailed template (pGL2CMV) but facing some serious problem. At this moment I am trying to standardize the method. I have PCR amplified the template and done oligo-dc-tailing of that template DNA. Agarose gel electrophoresis showed a little shift in the lane containing the tailed template indicating that the tailing reaction was successful. The expected length of my template is ~530nt. I am using HELA nuclear extract as the source of polII. I have started with a 20 μl reaction using 150ng of template and incubated the template with 20mM HEPES-KOH pH 7.6, 7 mM MgCl2, 100 μm NTPs(ATP, GTP and UTP), 10μm CTPs and 5 μ curie alpha-[32P]-CTP, ribolock and nuclear extract at 30 degree for 30 minutes. Sample is then processed (phenol: chloroform:isoamyl alcohol extraction) and run on 6% urea-PAGE. The gel is then dried and exposed to x ray film for desired time periods (from 30 mins to 48 hours at -80 degree). But I could not get any bands. Later I increased the incubation period upto 3 hours but still could not get any signal.                          I would be thankful if anyone of you give me some suggestions or comments and help me in troubleshooting it. Thank you.