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Differences in shape of melt curve

qPCR Melt curve

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#1 gmcleod

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Posted 06 June 2015 - 11:31 AM

I saw amplification in a negative control (not RT-, just not a sample where expression was expected) matches, or nearly matches, the melt curves for the expected product. But, there are very small differences in the shape/slope.

 

P14.png

Negatives = two blue curves.

 

P142.png

Negatives = two red curves with smallest peaks. Same primer pair as previous graph

 

nanog.png

Negatives = two blue curves with the highest peaks

 

[wishful thinking]: Would this be enough to rule them out as the product?

 

Thanks in advance for any info/advice...



#2 Mad Researcher

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Posted 06 June 2015 - 12:31 PM

I saw amplification in a negative control (not RT-, just not a sample where expression was expected) matches, or nearly matches, the melt curves for the expected product. But, there are very small differences in the shape/slope.

 

attachicon.gifP14.png

Negatives = two blue curves.

 

attachicon.gifP142.png

Negatives = two red curves with smallest peaks. Same primer pair as previous graph

 

attachicon.gifnanog.png

Negatives = two blue curves with the highest peaks

 

[wishful thinking]: Would this be enough to rule them out as the product?

 

Thanks in advance for any info/advice...

Hi,

 

Do you mean a non template control i.e. without any cDNA?


Cheers,

Mad Researcher

#3 gmcleod

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Posted 06 June 2015 - 03:43 PM

Hi, it's not a NTC either. It's a normal cDNA sample from cells that are not supposed to express the genes...

#4 Mad Researcher

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Posted 06 June 2015 - 11:36 PM

Perhaps a contamination in your reagents.

What about your positive and negative control?


Cheers,

Mad Researcher

#5 gmcleod

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Posted 07 June 2015 - 07:48 AM

Hmm, well, there was no amplification in any of the NTC or RT- controls; the positive ones are the other curves shown. 

 

I'm looking specifically at the differences in slope/shape at the beginning of the curves. Eg, the positives in the first and third pictures have a small shoulder before the peak, but not the negatives. Wouldn't the exact same product melt exactly the same way? Or is the difference so small that it doesn't matter?

 

Thanks again in advance







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