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
- - - - -

>100% qPCR efficiency


  • Please log in to reply
2 replies to this topic

#1 Kenyon Applebee

Kenyon Applebee

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 13 May 2011 - 06:00 PM

Hi there,

I am still getting a grip on all of the tricks and details of qPCR analysis. I keep seeing a shoulder on the left side of my melting curves and I have not been able to figure out whether this is normal (melting of random cDNA secondary structure) or a sign of primer dimers. I may have used too much cDNA in the run that produced the attached melting curve, but I would like to know if this could explain getting >100% efficiency (slopes of ~2.8 on log(cDNA dilution) vs Ct), or if I have primer dimers. None of my no-template controls had any peaks in the melt curve, which is why I haven't just gone ahead and designed new primers (plus two of these primers I got from another lab's publication, suggesting they work)

The melt curve includes data from a plate with three different primer pairs.

Thank you!

-Kenyon

Attached Thumbnails

  • MeltCurve-May13_11.PNG


#2 Trof

Trof

    Brain on a stick

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,201 posts
109
Excellent

Posted 14 May 2011 - 08:00 AM

Your melting curves look normal, the shoulder is always there. I don't see any dimers (but the lower temperatures are a bit obscured on the graph, to many curves). I don't get why you abandoned your first primers when you have no peaks in non-template control, there should be none.
Reason for >100% efficiency could be inhibition in higher concentrations, or I often see it in really short amplicons (like 60-70 bp). When it's not bigger than 110% you shouldn't worry about it IMHO.

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 dtae

dtae

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 59 posts
1
Neutral

Posted 17 May 2011 - 08:45 AM

To see whether the >100 E is due to standard curve artifacts you should consider calculating well-specific E values (e.g. with LinRegPCR if your software doesn't support it).

D




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

©1999-2013 Protocol Online, All rights reserved.