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Perfect dilution curve, but double peak melting curve on SYBR qPCR

qPCR SYBR melting curve

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7 replies to this topic

#1 Marvin

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Posted 06 June 2013 - 05:31 PM

Hi, All

I would really appreciate if you can give me some advice.

I'm using SYBR qPCR to amplify a fungal gene from genomic DNA. I've made a 1/10 dilution and the curve seems good, but the melting curve showed two peaks. I don't think it is primer dimers becuase there is noting on NTC (no amplificatin, no melting curve signal).


What else could it be? I'm amplified the ITS region (the barcode gene for fungi).

Many thanks.

Zewei

amplification curve:
Posted Image

melting curve:
Posted Image

#2 phage434

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Posted 06 June 2013 - 05:33 PM

That usually means you have two distinct products. Perhaps you have multiple species in your sample?

#3 Marvin

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Posted 06 June 2013 - 05:44 PM

That usually means you have two distinct products. Perhaps you have multiple species in your sample?


thanks.

But It doesn't look like the case. I extracted DNA from a pure petri dish culture and sequence the amplified region to verify it is pure culture. My primers were designed to only amplify the species I'm studyiing, at least in silico analysis (primer blast) showed it is unique.

I've also apply my assay on other fungi, which shouldn't be amplified, and they didn't.

#4 phage434

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Posted 06 June 2013 - 05:56 PM

Have you run the product on a gel to see if there are multiple bands?

#5 Marvin

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Posted 06 June 2013 - 05:59 PM

Have you run the product on a gel to see if there are multiple bands?


I'll do it tomorrow if they didn't throw off my plate Posted Image But I've run the product on gel using tranditional PCR this week using same primers, and only got a single band.

#6 phage434

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Posted 07 June 2013 - 05:16 AM

Even if there is a single band, it could be two sequences -- the ITS regions is typically the same length in different species. Only cloning and sequencing the fragments would really tell you if there are two different sequences.

#7 Marvin

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Posted 07 June 2013 - 02:01 PM

Even if there is a single band, it could be two sequences -- the ITS regions is typically the same length in different species. Only cloning and sequencing the fragments would really tell you if there are two different sequences.


That is true. Anyway I ran the gel today, 1X TAE, 1% agarose for 1hr, only saw a single sharp band.

I've sent the product for Sanger sequencing, will update it next week.

But I think one possibility is that I've got heterozygous products, since fungi are Dikarya.

#8 Hela

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Posted 02 October 2014 - 05:21 PM

Hi, I know this is a bit late answer to your post, but I was researching a similar problem today and here is what I found: you may still get 2 peaks on your melt curve while you amplify only one product, it is because of the way your amplicon melts, it may have some high GC region or so and part of it may melt easier while another not , and give rise to 2 peaks. Here is a link to an axample that depicts that:

https://www.idtdna.c...not-a-diagnosis

 

Try to run your amplicon on the Umelt melting curve prediction software,  free online tool created at the University of Utah  -if it predicts the 2 peaks you will have your explanation.







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