Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

Troubles with Fusion PCR - (Mar/28/2014 )

I am trying to make a fusion PCR product with a 5.5kb and a 720bp band. However, I am not getting anything after I anneal and elongate the products. I am using equimolar concentrations of both DNA products and I have an extension time of 6.5 min at 68C. Does anyone have any experience with fusing large fragments that could give me some pointers? I have tried adding more dNTPs and more Taq polymerase to the mixes, but that has not worked for me. 


Taq will struggle with generating products of that size, try a long read polymerase such as KOD or phusion.