Fusion PCR

[DeltaScience]

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

[CharonY]

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.

[DeltaScience]

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