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TaqMan real time PCR using abi 7700 system-troubleshooting


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#1 Jka83

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Posted 20 May 2009 - 12:28 AM

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 Im 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. Its 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

#2 Jka83

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Posted 23 May 2009 - 05:19 AM

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 Im 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. Its 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 Im worrying for nothing?

Thanks!

#3 tea-test

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Posted 23 May 2009 - 12:17 PM

here link in post #2 you will find a document where the correct baseline and treshold cycle settings on an ABI7700 are described.
tea-test: The artist formerly known as Ned Land

#4 Jka83

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Posted 25 May 2009 - 12:09 AM

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, Ive checked that document but it doesnt tell me the actual reason for this phenomenon.
Here, I attach the visualisation of the problem( or if maybe its 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



#5 tea-test

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Posted 25 May 2009 - 02:41 AM

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 :rolleyes:
tea-test: The artist formerly known as Ned Land

#6 Jka83

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Posted 25 May 2009 - 08:42 AM

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

Thats 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!




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