positive NTC in real time reaction - (Sep/03/2012 )
nicelady8 on Thu Sep 6 10:55:38 2012 said:
Yes, it is SYBR assay, and the melting curves are the same. As I know, dimer gives two picks, but my reaction has one. Maybe I need to replace my primers anyway.
Dimers give one peak in NTC, but what's important, their Tm is usually much lower, so you can tell. (when you have dimers and the product, then there are two peaks, but very common thing is non-template dimer, which only forms in the absence of template, it usually means you can take NTCs as negative)
If you checked also the length of the product on gel it is indeed contamination.
For that, it stays true what I wrote earlier. It's unlikely you've got contaminated reagents, because all NTCs would be positive. So.. cross contamination from pipetting, or contamination of well.
What I personally belive is that sometimes even it is not contamination or even if it is not primer-dimer, we can still see kind of amplification in the reaction. Which infact is not an amplification. This might be because of high amount of primer being used. Sometimes when we generate primer, it might be that it will never form the primer-dimer, for example: the primer designed by Cawthon for telomere length measurement using real-time PCR.
But, it still shows some amplification sometimes, its because of unused primer getting jumbled which migth somehow retain the SYBR green showing some kind of amplication, which is not real.
So, if you are sure about your techniques and every steps you have gone through, there is nothing to worry about the amplificaiton seen in the later cycles. Just like you mentioned above 28-cycles. Which is fairly acceptable.
Good luck with your research!
Have a great time ahead.
The option of the SYBR signal originating from unused primer is interesting, however we do see band on a gel in some of our NTCs that have a signal, but different melting profile. Also SYBR only binds double stranded DNA, so primers would need to be align somehow or form a secondary structures. From this is however only a small step to a dimer. Maybe all sorts of thing happends there.
But important is it only happens in the absence of template, and that it has a different melitng profile. I would certainly worry about amplification seen in later cycles if it had the same melting profile as a product, because 28 is not unusual value for some samples. It would not be acceptable, so it's important to add melting step after each amplification with SYBR, otherwise you don't know what exactly are you looking at in the amplification graph.