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Minimal total volume for real time - (Jul/09/2006 )

Hi everyone,

I have AP 7000 SDS machine in my lab and I've noted that the minimum volume recommended for a reaction is 25 ul. I was wondering if anyone out there had tried a volume below such recommendation? Say, about 10 ul to 15 ul?

Thanks & Cheers,

-I love MSGs!-

Hi there
I have done 18ul reactions and they have been fine. I know someone who is going to try 5ul reactions for a very large study in order to cut costs. I think it depends very much on how abundant your gene of interest is. If you start doing really low volumes you may not detect low abundance genes.
Hope this helps


I have routinely done qPCR in 12 and 10 total reaction volumes. Exceptionally, I have succeed in doing it in 5 ul for some targets...

Never trust reps for minimal volumes!

-erica arborea-

Hi guys,

Thanks for the assurance. Oh ya, the RT was fine even though I used decreased volume.

I heard from my colleague that different machines have different sensitivity. She advised me not to work on that way again, if possible. Haha...

-I love MSGs!-

Small volumes have poor reproducibility but if differense is 10 times or more and copy
number is lage it is OK