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,

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
Alison

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!

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...

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