Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

QPCR melting curves peaks


  • Please log in to reply
5 replies to this topic

#1 mamasaves

mamasaves

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 15 February 2011 - 05:09 PM

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.

AK Bio 3 4403 Melting Curve 020211.jpg

#2 Trof

Trof

    Brain on a stick

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,194 posts
106
Excellent

Posted 17 February 2011 - 01:39 AM

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.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#3 mamasaves

mamasaves

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 17 February 2011 - 08:35 AM

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.

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.



#4 Maddie

Maddie

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 289 posts
3
Neutral

Posted 17 February 2011 - 12:54 PM

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.


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?
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#5 UBClabbie

UBClabbie

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 65 posts
8
Neutral

Posted 17 February 2011 - 04:35 PM

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

#6 Trof

Trof

    Brain on a stick

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,194 posts
106
Excellent

Posted 21 February 2011 - 01:57 AM

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.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.