qRT-PCR effeciency

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Zelda
Zelda's picture
qRT-PCR effeciency

 Hi all
I have a problem with my standard curve. It is giving me a positive slop and negative effeciency percentage??
What might be the problem??/

Thank you

Ivan Delgado
Ivan Delgado's picture
Hi Zelda,

Hi Zelda,

Please post some data, and images, of your standard curve so that we can help you. Some things that would be useful to see include: 

1. A melt curve of your amplicon using SYBR Green (to make sure you are amplifying a PCR product and not simply detecting primer dimers

2. The data that gave you the positive slope (Ct values and their respective dilutions)

3. Your actual efficiency number. A negative efficiency does not exist, so it could be you are calculating your efficiency the wrong way (or your assay is simply not working). 

Cheers

Zelda
Zelda's picture
 Hi 

 Hi 
Thank you. my data
     log  1, 2, 3, and 4
    ct value   log ct 1 26.98, 28,, 28.5, 28.88 respectvly

Ivan Delgado
Ivan Delgado's picture
Hi Zelda,

Hi Zelda,

I do not understand what you mean by log 1, 2, 3 and 4.

What concentrations of DNA did you use for each of the dilutions you used for your standard curve? It should be something like: 100 ng, 1,000 ng, 10,000 ng, ...

Did you run a melt curve of your amplicon?

Zelda
Zelda's picture
 I ploted the log of the

 I ploted the log of the doncentration which are 1-2-3-4 (x axis) against ct value for the four dilution
Should I include the undiluted sample in the curve??

Ivan Delgado
Ivan Delgado's picture
I see. So you are saying that

I see. So you are saying that your concentrations were: 

10, 100, 1000, and 10,000

If that is the case, then your Ct values should be different by a value of 3.3. Instead your Ct values are only different by a value of less than 1. 

Without being able to see your data (melt curve analysis, amplification plots, etc) I cannot really know what is wrong with your assay.