Melting curve - SYBR Green PCR (Jan/25/2008 )

I'm experiencing horrendous problem with getting a single peak melting temperature. What I'd obtained so far is of multiple peaks. Contamination? Does the protocol affect it? Primer design?

Please help diagnose problem! Many thanks beforehand.

In Distress

First of all, the range of temperatures you are doing your melting curve at is not adequate, repeat doing the melting between 55-60C to 90-95C. I suspect that what you are seeing is very low intensity fluorescence (background), that appears as peaks as you don't have a proper "product peak" to compare (your graph finishes at 70C, your real product should be after that...).

Thanks for your kind replies.

In other words, I should do optimisation again.... am I going the right direction?

I've run using the same protocol on conventional PCR and ran on gel. There were bands produced. Hence the craziness when I didn't get any good results for Real-time. I'll heed your advice and let you know the results.

Thanks again.

I have done according to your advice. Attached is the melting temperature curve graph. Any other ways to improve it?

Thanks!

Urgently in need of help

OK, that's an improvement at least now you're seeing real results. Still, your PCR is not specific as it looks as if you're getting two products. The first peak could be primer dimers, though I doubt it. First thing to do, run your products in a gel, to discriminate between unspecific product and primer dimers.
Do you blast your primers, are they specific?
Can you be amplifying two splice variants (assuming this is RT-PCR)?
What amount of primers do you input in your PCR?

Anyway, by the look of it, I would suggest you redesign your primers...

OK, that's an improvement at least now you're seeing real results. Still, your PCR is not specific as it looks as if you're getting two products. The first peak could be primer dimers, though I doubt it. First thing to do, run your products in a gel, to discriminate between unspecific product and primer dimers.
Do you blast your primers, are they specific?
Can you be amplifying two splice variants (assuming this is RT-PCR)?
What amount of primers do you input in your PCR?

Anyway, by the look of it, I would suggest you redesign your primers...

Ummm... 0.28mM sound far too much to me.... Are you sure you didn't mean to say 0.28uM?