RT-PCR - RT_PCR (Jul/05/2005 )
i'm going to do RT-PCR on the mRNA samples. i would like to have an idea what should i do next, after RT_PCR? should i directly clone into the expression vectors or i need to put the clones first into the plasmid form? what is the best simplest way . please give me some ideas.
After you run you RT-PCR, you run a gel to make sure the presence of your specific band. If you have primer dimer in you reaction you run a PCR clearn up. If you have unspecific band in your PCR product, cut the specific band a run gel purification.
Later, identify common RE site which present in both your PCR product or plasmid (multiple cloning site). Perform RE digestion on both of your PCR product and plasid. Follow by ligation and transfection of competent cell.
After RT, i suggest that you clone it into a TA vector. Then sequence it to confirm that it is your gene of interest. Then you you can subclone it into an express vector.