I have the viral gene inserted in the pGEM plasmid (done by Genscript). I am trying to set up the standard curve for the real time PCR detection of the virus. So, I cut the plasmid with SAC 1 to linearize it and then I gel extracted the linear plasmid. This linear plasmid I used as template for in vitro transcription with T7 ribomax kit. And at this step I am stuck. The control of the kit did not work, so I got new kit. Then the plasmid gave me multiple RNA bands. Then I also used Klenow but there was no difference when I used the Klenow. Now again, the kit does not work with either the control or the sample. I am following all the good laboratory practices and still I have lot of problems for 2 months with this kit. Can anyone suggest me some tips or a better kit. Thanks!
- really frustrated !!