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?

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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!

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I think small volume reaction (<20ul) work, but variation will be increase...

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A bio-rad rep told us that people are using 10ul reactions, but I havn't seen it.

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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.

Del.

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