Amplimer length for TaqMan - (Aug/07/2007 )

Hi!

I have heard that the optimal length for a real-time PCR product is between 50 and 200 bp. I wonder why you couldn´t have longer amplicons?

I have seen answers on this side that as a longer amplicon consumes more reagents as dNTP the exponential phase becomes shorter and that is one reason. If you mix your own mastermix, then you could add more dNTP and enyme. Will this still be a problem?

Is it a difference between the length preference if you use SYBRgreen or a probe?

I would agree that you _could_ use larger amplicons (200+ nts), however you would have to scale up the dNTPs, and SYBR concentration. Also, make sure you don't skip a No-RT and the No-Template controls. Also, keep in mind that the melting point of larger amplicons is going to be much higher and have a broader peak; which may be a problem esp. if you are using SYBR as your reporter dye.