5 replies to this topic

[mamasaves]

Hello,

In a couple of my experiments I see that my standard curve has a single peak in the melting curve and my samples have another.  They are fairly close, but it is still worrisome.  I am attaching a photo of one of the more extreme cases.  All of course are using the same primer pair, so I am a little perplexed that the products being amplified have different melting temperatures.  Please advise.

[Trof]

Do you use SYBR Green? What type of experiment is it, absolute or relative quantification, I mean, do you use standards from some kit or you just dilute your abundant sample to create a standard curve?
There may be slight variation between Tm, but I'm not sure if that profound. The best way to check what exactly are you amplifying is check the products on gel (just take them from the completed run and pipet to EtBr gel) to see a same-sized band, or better to sequence it. Maybe the sequence of standards and samples differs in some SNP or something, but now that I think of it, it doesn't seem much likely.

[mamasaves]

I am using SYBR Green and I am using the absolute method to create a standard curve.

I was planning on running some of them on a gel to check, but I just wanted to make sure I wasn't missing something completely obvious.  Thanks for your response.

Trof, on 17 February 2011 - 01:39 AM, said:

Do you use SYBR Green? What type of experiment is it, absolute or relative quantification, I mean, do you use standards from some kit or you just dilute your abundant sample to create a standard curve?
There may be slight variation between Tm, but I'm not sure if that profound. The best way to check what exactly are you amplifying is check the products on gel (just take them from the completed run and pipet to EtBr gel) to see a same-sized band, or better to sequence it. Maybe the sequence of standards and samples differs in some SNP or something, but now that I think of it, it doesn't seem much likely.

[Maddie]

mamasaves, on 17 February 2011 - 08:35 AM, said:

I am using SYBR Green and I am using the absolute method to create a standard curve.

I was planning on running some of them on a gel to check, but I just wanted to make sure I wasn't missing something completely obvious.  Thanks for your response.

Trof, on 17 February 2011 - 01:39 AM, said:

Do you use SYBR Green? What type of experiment is it, absolute or relative quantification, I mean, do you use standards from some kit or you just dilute your abundant sample to create a standard curve?
There may be slight variation between Tm, but I'm not sure if that profound. The best way to check what exactly are you amplifying is check the products on gel (just take them from the completed run and pipet to EtBr gel) to see a same-sized band, or better to sequence it. Maybe the sequence of standards and samples differs in some SNP or something, but now that I think of it, it doesn't seem much likely.

Would it work to run the amplification product on an agarose gel? I mean if there is already SYBGreen between the DNA strands, I'm guessing Ethidium bromide can't intercalate. Or maybe run this on a gel without EthBro? Just wondering...

So, what you're saying is that your standards and your samples contain DNA from the same species, right? Only 1?

[UBClabbie]

I've never had a program running samples on a gel after amplifying using qPCR. I think each dye binds to DNA differently.

[Trof]

Maddie, on 17 February 2011 - 12:54 PM, said:

Would it work to run the amplification product on an agarose gel? I mean if there is already SYBGreen between the DNA strands, I'm guessing Ethidium bromide can't intercalate. Or maybe run this on a gel without EthBro? Just wondering...

I wondered also in the beginning, but we did it several times and it worked.

Edited by Trof, 21 February 2011 - 01:58 AM.