problems with multiplex PCR

[Silvia_84]

Hi all,

 

I tried to do multiplex PCR to amplify more genes at once with the biomix red polymerase. Unfortunately it did not work (no bands at all, not even primer dimers)and I have no idea of what goes wrong. The primers should be ok because I am using those already used by another group who has already done the same multiplex PCR.I don't ' know if I make some mistake in the procedure:

 

For a 25 ul of reaction mixture I added:

 

12.5 ul of BIOMIX RED

1 ul of each primer (they are in total 8 because the genes I am amplifying 4 genes together)

3.5 ul of water

 

The single PCR, done with the primers separately is fine.. so there must be something wrong in the multiplex set-up

 

Thank you in advance.