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Help: Linear Amplification Using Hydrolysis Probes

linear amplification qpcr real time

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3 replies to this topic

#1 bronson3000

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Posted 01 November 2011 - 10:39 AM

Hi guys,

I have looked all over the internet for a similar problem but cannot seem to find a discussion addressing it.

I am currently trying to get qPCR assays working for three genes, right now I am using leaf-extracted genomic DNA. My primers proved to work well when I tested them using conventional PCR, and I tried out real time with SYBR green which also worked well. However, for my project I need to use probes (Taq-Man type), which were designed using software incorporating my primer design. I have tried four runs with these probes and each time (for all three genes) I get totally linear amplification. Wondering if anybody has ever run into this or would have suggestions? I've attached a couple of plots, the green sigmoidal curves are using SYBR and the dark blue lines are using my probes.

Thanks!

Attached Thumbnails

  • real time Oct 3.jpg
  • real time sept 6.jpg


#2 leelee

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Posted 01 November 2011 - 06:00 PM

I'm not very experienced with qpcr (although I am using probes based assays at the moment), but my guess would be that what you are seeing is not amplification but rather degradation of the probe, where the fluor is being cleaved/released from your probe.

Have you titrated the amount of probe you are using?
Are you planning on multiplexing? I assume you are testing each probe individually first?

#3 Archie

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Posted 02 November 2011 - 03:49 AM

Could you attach log view of the plots as well?

As said above check for probe degradation: run a normal reaction but replace the MasterMix with water to see if there is autofluorescence coming from a degraded probe.

I'm not sure if you get actual amplification with the hydrolysis probe. Have you tested the primer-probe combination with other (more pure) samples?
How did you design, align etc. the probe?

#4 bronson3000

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Posted 02 November 2011 - 09:35 AM

Thanks for the replies!

I designed the primer/probe combinations using IDT's PrimerQuest, which is where I ordered them from as well. I also checked the primers in Primer3 although I believe PrimerQuest is based off of Primer3 anyway. I have only tried the probes with DNA extracted from leaves but from multiple occasions, also it was extracted using a kit and the DNA each time was subjected to spec analysis first, showing good quality and quantity. I haven't checked if it's a degradation issue.. that's a good suggestion though. I've attached the log views of the plots.

Thanks again.

Attached Thumbnails

  • Log Oct 3 Run.jpg
  • Log Sept 6 Run.jpg






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