2 replies to this topic

[neuropath]

Hi,

I am planning to run semi-quantitative real time pcr assays in 96-well plates on a Roche Lightcycler 480. The problem is, I have limited amount of RNA sample but I have many genes to assay for. So in order to complete the experiment with what little RNA that I have, I am thinking of scaling down the volume of my PCR reactions. I am using the Bio-Rad Ssofast Evagreen Supermix reagent, which recommends 20ul reaction volume. Has anyone ever tried lower volumes on the Lightcycler, like 10 ul for example? I guess I can do a trial run with controls to test it but I just want to hear from someone with first-hand experience first.

Any advice is appreciated

Thanks.

Neuropath

[Trof]

We run 10ul reactions on LC480 on regular basis. With a Roche Mastermix.

If you have a limited starting amount of cDNA you can also do a preamplification, I think ABI offers such kit.

[neuropath]

Thanks Trof. That's great information