5 replies to this topic

[littleaxt]

Hi all!

I am about to test primers, a never ending story....
I tested several primers and I always thought, and this is what I had experienced so far, that the dimers they might produce also occur in my no template control. But this time they did not. And this not only in one primer system.
The only difference between those lines is the added cDNA (see attached picture, lower line is the NTC, two peaks are cDNA). Could there be something else in the cDNA that might interfere in the reaction and give such a strong SYBR green signal? Or might just be sample or run specific?

Cheers!

Jan

Attached Thumbnails

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[pcrman]

I don't quite understand what your problem is. Regarding your question in the title, yes, primer dimers also appear in no template controls

[littleaxt]

Sorry for not making it clear, but if you take a look at the picture, the lower line is my NTC and it seems primer dimers did not occur in it. So I was confused and thought the unspecific fluorescence I observed in the sample could be due to something else than primer dimers. It did not only occur in one sample but in three or four otherwise I would not have asked here.
Any idea what else in the cDNA could cause this unspecific fluorescence?

pcrman, on May 19 2009, 10:23 PM, said:

I don't quite understand what your problem is. Regarding your question in the title, yes, primer dimers also appear in no template controls

[almost a doctor]

littleaxt, on May 20 2009, 07:42 AM, said:

Sorry for not making it clear, but if you take a look at the picture, the lower line is my NTC and it seems primer dimers did not occur in it. So I was confused and thought the unspecific fluorescence I observed in the sample could be due to something else than primer dimers. It did not only occur in one sample but in three or four otherwise I would not have asked here.
Any idea what else in the cDNA could cause this unspecific fluorescence?

pcrman, on May 19 2009, 10:23 PM, said:

I don't quite understand what your problem is. Regarding your question in the title, yes, primer dimers also appear in no template controls

Hi, I don't see your problem here, there are no primer dimers in your sample.  
You are looking at the derivative plot of your data so it might be a bit confusing as the peak is not fluorescence but actually lack of it.  If you look at your raw data you might see it better. In your NTC samples you should have no DNA at all (other than primer dimer which I believe you dont have), so your NTC plot should look the same in the derivative or raw form. However if you look at your samples in raw data you'll see that the fluorescence (due to the presence of your product) will decrease with temperature until ir reaches ~0 at the Tm (what you see as a peak in the derivative plot). What you are seeing as "background" is just normal and caused by the presence of product, if you had primer dimer you will have a proper peak (similar to that for your product) at a lower Tm (@70-75C)

Hope this helps and makes sense. Any other question ask  

[swanny]

Have you run your samples on a gel?

[littleaxt]

swanny, on May 21 2009, 07:11 AM, said:

Have you run your samples on a gel?

No, I haven't yet.

But I guess these problems are just caused by the bad performance of the PCR. To some reasons I had very low fluorescence signal anyway and by this the normal background seemed to be enormous high. Just wait for my fresh ordered SYBR green, as as it arrives I will try to optimze the reaction and see what happens.

Thanks to all that tried to help!