Volume of Real time PCR mastermix and reaction - (Feb/13/2006 )
in the coming days, I think I need to do a large no., of reaction of Real time PCR, I have used the ABI7700 and ABI mastermix and normally use 25ul for 1 reaction. However, as u know, it spends a lot of money and so I would like to ask what is the minimum vol. of reaction u have used and achieve a good result also?
An ABI representative told me that the smallest volume they recommend is 20 µL. But of course, their opinion could be biased because they want to sell their product!
I think small volume reaction (<20ul) work, but variation will be increase...
A bio-rad rep told us that people are using 10ul reactions, but I havn't seen it.
We routinely use 10ul reactions (8ul mastermix + 2ul template) on an ABI7000. This works fine, but you should be aware that smaller volumes will give you a shorter pathlength for detection, so you lose some sensitivity.