improve the DNA cons. in RT-PCR - (Jan/11/2007 )
I am doing RT-PCR in order to do sequencing. The problem is that I am getting to small bands on my gel. (I am not sure that it is enough DNA for sequencing).
My primers Tm is 62c and the annealing temp. is 57c. I am doing 35 cycles.
What should I do in order to improve the amount of DNA that I get in the gel?
Lower your annealing temp in the last 20-25 cyles by 1 or 2°C.
Perform an extra PCR to get enough product to get your sequence right?
it's better clone the PCR product first and amplify your PCR product by bacterium