Product detectible on gel, melting curve peak, but no amplification curve

[emucaki]

Hi guys,

I've ran into a bit of an oddity this week. I have a SYBR Green assay where a 110bp product being made from cDNA I've used for other purposes and had no trouble with. However, I keep getting no product in my amplification plot (while other primer sets in the same assay are giving a signal). But there is seemingly a product being formed when I look at the melting curve (peaking at around 1400 RFUs).

I've also run these product using regular Taq (different thermocycler) and the product has been visible on a gel. Anyone have any ideas why this would be happening?

EDIT - this is on a Stratagene Mx3005P if that matters

[Aga]

Hi emucaki. I need to know one more thing. Do you perform multiplex reaction?

[emucaki]

Hi, it is a simple SYBR Green reaction with one primer set. It is so strange because there is no amplification on the qPCR but the product is right there.

Right now I am repeating the experiment with a free sample Eva Green kit, maybe there's something strange about my product that isn't allowing SYBR to get in there. It isn't the machine, and it isn't the enzyme. I'll edit in the results once it's done.

- EDIT - it fixed the problem. For some reason our SYBR kit is not working well with this amplification. The Eva Green kit produced an amplification plot that looked normally logarithmic. The sequence is GC rich, but nothing stands out into why SYBR seems to have a hard time getting in (or what ever else could be the reason for this problem).

[Aga]

I can only think of one mechanism leading to this problem.
Sometimes it's the issue with your DNA concentration. Some kits with SYBR Green are designed so that the maximum template concentration is e.g. 100ng per 20ul reaction. This depends on SYBR Green concentration - if it's too low for the template, it saturates all the available dsDNA at the beginning and there is not enough dye to show exponential amplification of your product. The product could be seen on the gel or even melting curve though, cause polymerase did its work.

[here444]

cool, this is good to know