need advice on qPCR results (see attachment) - melting curve - gel picture (Oct/23/2006 )

hi,

i have trouble with the melting curve a specific amplicon.
die melting curve looks really fuzzy, however on the gel,
i see only single bands. the duplicate reactions also come up
equally. would you use thes data for quantification?
how can it be that the gel looks nice, but the melting curve
looks that fuzzy?
i really appreciate your help

kind regards,

martin

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Dear Martin,
What is your PCR profile and how did you performed your melting curve analysis?
Could you please attach your amplification curve as well?

Thank you

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hi hadrian,

so the amplification curves are in the attached pdf file.

i am using the takara ExTAQ SybrGreen qPCR premix in an opticon2 (MJ) cycler,
45x 15 s 94°C
20 s 58°C
20 s 72°C

melting curve is done from 55 to 95° C reading every 0.2°C.

the thing is that all the other amplicons show a really sharp and nice melting curve plus a nice single band on the gel, but with
this one amplicon (attachment) i get this strange melting curves and i don't have an explaination for it.

cheers

martin

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Is it me, or does it look like you do have dimers on your gel?

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I think it looks very much like a problem with primer-dimers; I totally agree with tap14

further, it is good to show the y-axis scale on the melting-curve plot if you can...but that is irrespective of the dimer issue

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Hello Martin,
It is not surprising that you only see one band on your gels. This is due to the lack of sensitivity and separation characteristics of a gel. The melt curve indicates that you have some non-specific amplification and you really should re-design your primers.

hi,

i have trouble with the melting curve a specific amplicon.
die melting curve looks really fuzzy, however on the gel,
i see only single bands. the duplicate reactions also come up
equally. would you use thes data for quantification?
how can it be that the gel looks nice, but the melting curve
looks that fuzzy?
i really appreciate your help

kind regards,

martin

[attachment=2014:attachment]
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