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Strange amplification plots with high Ct variability


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#1 Greg2013

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Posted 15 August 2013 - 01:06 PM

Hi,

 

Can anyone help me interpret this raw data amplification plot? Why do my 18S plots seem so flat, i.e. not sigmoidal? Why are my Ct values for each technical replicate so variable?  I can't seem to figure out what I'm doing wrong. Blue are my NTC's. My 18S primer set seems to be a little dirty.  Melt curve analysis shows single amplification products. Chemistry is SYBR green on an Eppendorf Realplex2.

 

Rxns are as follows:

10 uL 2x SYBR

0.5 uL 10uM For

0.5 uL 10uM Rev

7.0 uL Nuclease free H2O

2.0 uL 1:25 cDNA Template --> cDNA dilution: 1 ug total RNA in 20 uL RT rxn; 2 uL cDNA mix + 48 uL ddH20

 

Let me know if any other information is needed.

 

THANKS!!

Attached Thumbnails

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  • all.jpg
  • PCNA.jpg


#2 traveler9907

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Posted 29 August 2013 - 07:25 PM

Can you show us the log view? I don't see the 1st derivative melt profiles either. Usually it's the primers and/or samp,es that cause issues. Better reagents can be,p too. Check out www.bio-rad.com/supermixes_tutorial. Very useful for understanding qPCR performance.




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