Best product size for real-time RT-PCR - (Apr/03/2006 )

Dear Colleagues,

What is the best product size for RT-PCR? Do you think if I can do the product of 300bp with SYBR Green? I heard it is usually less than 100bp...

Thanks
Hy

For a real time PCR with SYBR green, PCR products should in arrange of 150~80bp. Longer product might work, but consume PCR component more quickly than short one, result in shorting the range of exponential phase in amplification, anyway, a 300bp products seems still acceptable for real time PCR with SYBR green.

Hi !

I can agree with rye. Optimal amplicon size in RealTime PCR is about 150bp, although new kits can handle products as large as 400bp (FullVelocity SYBR Green - Stratagene, DyNamo SYBR Green - Finnezymes). I had a 350bp amplicon and results were just fine. But still you have to keep in mind that larger products may strongly affect efficiency of RealTime.

Good Luck

[/quote]

Hi guys!
Do ya'll think the larger the product size the less volumes we should put in a reaction?


[/quote]

Dear Chris,
I don't thinks product size has any thing to do with reaction volume.