Hello everyone, well I'm performing some experiments using a new type of vector and for these I have to use Fusion PCR technique to get my genomic fragment into the vector, but I have been having troubles with these because my only Polymerase is the normal TaqPol haha, I think my problem lies in the Termocycler programing wich to be honest I dont know for this kind of PCR, I would really appreciate if someone could tell me the conditions needed for Fusion PCR with TaqPol, or some suggestions.

 

Thank you.

 

Greets

The conditions are largely dependent on your sequence. Also the T-overhang can cause problems. If you need troubleshooting help, you should post what precisely you are doing and what worked/did not work.

 

Fusion PCR is basically the same as an earlier technique named Gene SOEing published in Biotechniques, btw.

Well I was using a special poylmerase to get a blunt end PCR product to use in the fusion PCR, the Takara PrimeStar wich is awesome, but then i run out of polymerase heheh and so far I have managed to get good amplification for my PCR product but only faint bands of my Fusion Product and then the transformation wasnt working, so I have the PCR product with blunt ends but now I need to fuse it with my vector wich is specially designed to fuse with the first primers I used for the PCR products, but this using the TaqPol hehe, thats my problem because i dont know if the extension time and final extension could be the problem with my reaction.

 

Thanks