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qPCR amplification


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4 replies to this topic

#1 Angeline

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Posted 24 March 2014 - 05:24 AM

hi.

I'm wondering, is it possible to have nice amplification plot in qPCR but no band observable when the pcr product is run on etbr stained gel?



#2 Tabaluga

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Posted 24 March 2014 - 05:44 AM

How long is the expected bp length of the product ? Did your gel have an appropriate resolution and running time ?


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Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#3 Trof

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Posted 24 March 2014 - 06:55 AM

It's very possible to have nice amplification plot (usually high Cts) of primer-dimers.

These are really small and can just run out on ordinary gel, higher percentage gels are needed and mostly the dimers are visible as a thick smear.


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#4 Angeline

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Posted 25 March 2014 - 02:05 AM

I understand that there is a possibility of primer dimers if the ct is high.

But, is it possible to not get band on etbr stained 1.5% agarose gel if the ct value falls within the range of 8 to 15? 



#5 Trof

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Posted 25 March 2014 - 07:36 AM

Are you sure that your gel works? (or if you don't repeat it, that it worked at that moment?)

Can you run other "working" real-time PCR side by side and put both also on gel?

 

Did you do a melting curve analysis of that product?


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





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