real time PCR reaction volume

[aniahawaii]

Aloha to you All from Hawaii,
I am fairly new to qPCR word. I have two basic question

I am using Rotor gene 6000 for my qPCR (this machine takes 0.1ml and 0.2 tubes)

Does the qPCR reaction volume (negatively) influence the outcome? Can I use as little as 5µl or 10µl reaction? Or do I have to use 20µl?

Would air bubble trapped in a tube (mostly on the bottom as results of pipetting) negatively influence reaction/ reading?

Thanks for your help
A

[Aga]

Yes, air bubbles will affect the reading and results. However, they can occur ever with 20ul reaction volume. Can you centrifuge your reaction tubes? This would solve the problem.

You can go down to 10ul or lower, after optimisation of your reaction. If you do a specific assay for the first time I recommend 20ul, but later you can try lower volumes and compare the results with previous reaction in 20ul. With duplex/multiplex assays lower volumes may not be successful.

[amicia]

If You are using Rotor gene 6000, the centrifugation step is not necessary cause the system centrifuge the samples during the reaction

[aniahawaii]

Thank you for your input