Need Help! Weird melting curve - (Dec/27/2005 )

I have something that need to consult all your experience, i had run several of real time PCR, and I obtained some weird melting curve for my PPAR1 and PPAR2 gene. I attached the document for your reference and please comment on the melting curve and what can i do to correct the melting curve. Thank you.

hmmmm....need more information to be sure, but I have a couple of possibilities for you.

1st, have you run a gel to check your product?

2nd, how are the melting curves supposed to look? what are the predicted Tm's of your PPAR1 and PPAR2? did you get it to work in the past, and just now it is crapping out on you?

I can see that you are getting multiple products. Based on how you answered these questions, we can find a source of the problem. It could be a mix of products, like two amplicons with similar melting points...this could be due to poor primer design. Also, it could be a mix of product and primer-dimer; again, poor primer design. the hardest part of real-time is getting the damn primers right. another possibility is contamination of some sort; how are your negative controls? what about your -RT control? could be it be gDNA is giving you the other product?

When I have seen such curves in the past, my primer aliquots were degrading (I had resuspended them in depc-H2O and stored them in aliquots at -20 for about 6 months) ...it took me forever to realize the source, I had tried everything else because I thought it was a contaminant

Thanks for your reply, i checked the product on 1% agarose gel.. There are only 1 band per lane.

This is a new experiment, melting curve is still unknown yet.

Today i run another run of real time PCR, and found out that the primer concentration was too high (1uM) in the last PCR. I run with lower primer concentration (0.25 uM) and it has only 1 peak only, but the peak still have broad base.

Question: does width of the base of the melting curve peak represent the purity of the amplicon product?

how wide are you talking? those pix from before, that was not one product...multiple peaks and such on those two amplicons

I would recommend maybe trying a gradient of primer concentration, then running a gel of the products? I know it's a pain but you need to make sure you are getting the right thing (and by the way, if you know your sequence you can predict your amplicon Tm and it is a good idea to know what to expect!) you need to be sure you are not just getting primer-dimer...I am assuming you use SYBR green?


how tight is your 'one band'? the wideness/multiple peaks, to me, would suggest that you would probably see a little bit of a smear. did you run the gel out long enough to see for sure? and what was the size in comparison with what you expected to get? if your amplicon is around 100-150bp, you might want to up the % of your agarose to 1.5, 2ish and run it out for awhile. it may be that you are getting two products pretty close in size...

anyways, I don't know if this is helpful but I hope you can get it straightened out

Thank you for you advise, it had help me alot and now i think i got better experimental result.