Hi,

I am generating a ssDNA overhang from my PCR amplification. The length of the PCR product is ~160bp. In the forward primer, the first 20nt is regular DNA and the next 6 nt is pt modified. In the reverse primer, the first 6 nt is pt modified. In theory, this will give me a 140bp dsdna + a 20nt ssdna overhang. In reality, my T7 is not working on this subtrate while it is working in a control with no modified nt at all. Anybody has any idea what step is wrong in my design?

Thanks!