5 replies to this topic

[Jka83]

Hey guys

My problem is following. I have set up a real time PCR systeem for detection of this specific virus. Well, the primers and probe seem to work and I´m detecting the virus.
It is just this detail that bothers me.  When I look at my amplification plot in linear view, the reactions should flat at zero (the fluorescence should be flat at zero). That is not the case, the reactions sort of "fluctuate" around zero. It´s probably because of my baseline fluorescence is too high...Why is that? Any suggestions? Ever faced this kind of a problem?

Thank you in advance!

Cheers

[Jka83]

Jka83, on May 20 2009, 07:28 PM, said:

Hey guys

My problem is following. I have set up a real time PCR systeem for detection of this specific virus. Well, the primers and probe seem to work and I´m detecting the virus.
It is just this detail that bothers me.  When I look at my amplification plot in linear view, the reactions should flat at zero (the fluorescence should be flat at zero). That is not the case, the reactions sort of "fluctuate" around zero. It´s probably because of my baseline fluorescence is too high...Why is that? Any suggestions? Ever faced this kind of a problem?

Thank you in advance!

Cheers

Hey again

I would very much appreciate if someone would figure it out. Or tell if I´m worrying for nothing?

Thanks!

[tea-test]

here link in post #2 you will find a document where the correct baseline and treshold cycle settings on an ABI7700 are described.

[Jka83]

tea-test, on May 24 2009, 06:17 AM, said:

here link in post #2 you will find a document where the correct baseline and treshold cycle settings on an ABI7700 are described.

Hey

Yeah, I´ve checked that document but it doesn´t tell me the actual reason for this phenomenon.
Here, I attach the visualisation of the problem( or if maybe it´s not a problem after all?)

Pic 1: How my ampl.plot looks like
Pic 2: How it apparently should look like (according to ABI 7700 tutorial guide)

Cheers
Jka

Attached Files

•  ampl.plot_baseline_fluctuation.doc   237.5K   132 downloads

[tea-test]

I would say that there is no problem at all, your baseline fluorescence fluctuates a little bit but thats normal. Pic2 looks nicer but also the scale of the y-axis is different

[Jka83]

tea-test, on May 25 2009, 09:41 PM, said:

I would say that there is no problem at all, your baseline fluorescence fluctuates a little bit but thats normal. Pic2 looks nicer but also the scale of the y-axis is different

Hey

That´s probably the case.
Btw, do you happen to have any good tips for making the assay more sensitive? RT-time, probe concentration, something else?

Thank you!