Real Time: One Melting Curve 2 gel bands - (May/19/2006 )
In my real time experiment using the Roche Lightcyler and fast start DNA SYBR Green Master Mix I had one single melting peak at 85 degrees C (duplicate samples produced overlappig curves). The NTC capillary did not amplify any product and the NTC melting curve appears as a straight line. It looked to me like all was well....However, I ran a 1% agarose gel and there were two distinct bands around were I would expect my amplicon to be (~113 bp, GC% 48). The NTC capillary was completely clear on the gel (no bands). I waited several hours to run the PCR product on the gel...Any thoughts on what could have happened...I am still very new to this and would appreciate any thoughts.
Please attache a picture of your gel,
it's possible that I have the same problem. In many of my experiments the positive samples of a certain amplification system show a light band of the double size. The appearance seems to depend on the time the capillaries are stored before the product is run on a gel. It's possible that this is an artefact from secondary DNA structures.
I suggest you to check in your real rime pcr program in what temperature it start to do the melting curve. It could be that the melting point of the second band is very low so you can’t see it. If its like that I don’t think you have to be worry about it because in the point of time its read the florescence this band is not exist any more.
I don't know why you are surprised that two different sequences can have the same melting temperature. All this means is that they have similar GC content. Think about it this way: there are a zillion sequences, and only a few hundred distinguishable Tm's.
Thanks for all the replies. It appears I just needed to crankl up the annealing temperature even more.
Before the era of Applied Biosystems PDAR's we used to design our own primer, primer probe sets. The way we confirmed these PPP sets were working was by cloning the amplicon (usually in a run without probe and just primers) into the TOPO TA vector and picking up 5-10 sequences to make sure there was only the correct amplicon (be aware you will get some sequences which are just primer dimers). I suggest trying this as well. I have been to a few talks where they present work and show this lovely single peak for the melting point but I am always dubious that using this to conclude you are amplifying only your target is sufficient.
it can be SYBR artifact