Gain! - (May/01/2009 )

Hello all,

I'm using RotorGene 6000 for my qPCR applications. I've been using the machine for some time now but my amplification curves do not go higher than 10% fluorescence.

I think I'm adding all the reagents correctly but don't know why fluorescence level doesn't go higher that 10-11?

my gain for SYBR Green is at 5 now. should I increase it?......my curves start to appear from 20-25 cycles above.

Hi Curtis,

I'm using a rotorgene 3000 so not sure if this can help..
Last year I was told to use the auto gain calibration, ticking the option "auto gain calibration performed before first acquisition " setting up the temperature of acquisition at 72°C (the extension temp). the minimum and maximum fluorescence reading were set at 1 and 3 respectively..
Don't know if you can follow what I'm saying because I know they changed the software a bit between the 3000 and the 6000 machines.

As few months have gone since your post, I'd like to know if you could fix the problem in the meantime.

Cheers

Raffaella

Hi, thanks for your reply.

I put it on 7 now, it's working better but still the curves do not go very high. my friends say it could be because of my target sequence or the SYBR green that I'm using. But I'm fine now

My curves used to get to 100, with gain chaniching time to time as it was automatically set by the machine, now they barely make it up to 15... Don't know what's going on
I'm happy it's working for you tough..
Raffaella

Hi

I am using the 6000 and we often have to use the gain optimization function especially between new probe orders. I would recommend using the optimization as a change in value of 1 unit could make all the difference between a good run and a great run.

Best of Luck