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real time pcr melt curve and primer efficiency problem


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#1 krystle

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Posted 15 April 2010 - 11:55 PM

hi guys, i recently started running quite a bit of real time pcr, using primers passed down from seniors. I've used the primers for numerous experiments before being introduced to primer efficiency by another friend. So i tried measuring the primer efficiency and i realised that a few of them only have the efficiency of 70+%. Some of them are 90-105%, which i assume is okay. So my question is, do i really have to forsake the primers with 70+% efficiency and redesign them? Or is there some mathematical formula that i can use to account for the lower efficiency? Its really frustrating if i have to redesign and re-run them coz i've used them for so many experiments!


Another question, i realised that afew of the primers have some weird looking melting curve, even if the primer efficiency is quite good. I've attached a few photos. Does anyone know whats causing the front part of the curve to be so high? I know its not dimer right, coz i read that curve due to primer dimer should also be a sharp curve.

Thanks a lot for your help.

Attached Thumbnails

  • PU1_Melt_Curve.jpg
  • bactin_Melt_Curve.jpg


#2 chrisbelle

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Posted 22 April 2010 - 03:05 AM

hi guys, i recently started running quite a bit of real time pcr, using primers passed down from seniors. I've used the primers for numerous experiments before being introduced to primer efficiency by another friend. So i tried measuring the primer efficiency and i realised that a few of them only have the efficiency of 70+%. Some of them are 90-105%, which i assume is okay. So my question is, do i really have to forsake the primers with 70+% efficiency and redesign them? Or is there some mathematical formula that i can use to account for the lower efficiency? Its really frustrating if i have to redesign and re-run them coz i've used them for so many experiments!


Another question, i realised that afew of the primers have some weird looking melting curve, even if the primer efficiency is quite good. I've attached a few photos. Does anyone know whats causing the front part of the curve to be so high? I know its not dimer right, coz i read that curve due to primer dimer should also be a sharp curve.

Thanks a lot for your help.


70+ is quite ok if you are doing relative quantitation. use a calculation method that would take the efficiency into account when calcating gene expression, eg Pfaffl method. for your second q, try reducing primer concentration. its not necessary for primer dimers to be a sharp curve.

Chris
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#3 krystle

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Posted 25 April 2010 - 09:46 PM

hi guys, i recently started running quite a bit of real time pcr, using primers passed down from seniors. I've used the primers for numerous experiments before being introduced to primer efficiency by another friend. So i tried measuring the primer efficiency and i realised that a few of them only have the efficiency of 70+%. Some of them are 90-105%, which i assume is okay. So my question is, do i really have to forsake the primers with 70+% efficiency and redesign them? Or is there some mathematical formula that i can use to account for the lower efficiency? Its really frustrating if i have to redesign and re-run them coz i've used them for so many experiments!


Another question, i realised that afew of the primers have some weird looking melting curve, even if the primer efficiency is quite good. I've attached a few photos. Does anyone know whats causing the front part of the curve to be so high? I know its not dimer right, coz i read that curve due to primer dimer should also be a sharp curve.

Thanks a lot for your help.


70+ is quite ok if you are doing relative quantitation. use a calculation method that would take the efficiency into account when calcating gene expression, eg Pfaffl method. for your second q, try reducing primer concentration. its not necessary for primer dimers to be a sharp curve.

Chris



Hi, thank you for your reply. If i reduce the primer concentration, does it mean i have to redo the calculation of the primer efficiency since the reaction condition is now slightly different?

#4 sarahmartin

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Posted 22 November 2010 - 08:12 PM

Have you managed to fix this issue? I'm having similar problems. I use the Applied Biosystems StepOnePlus instrument and their Power SYBR Green mix. We have only just started doing real-time in the lab so all the primers we have are new but either gained from databases or commercially available ones.
I also get a shoulder at the start of my melt curve though not quite as large. I've tried diluting out my primers but this also results in much smaller deltaRn - is that a problem? Now even with the commercial primers they have a peak too. I know I had primer dimers with my original primers because i could see them on the gel. But primer dimers aren't visible with the new primers yet the shoulder is still there? Each run I do I get different amplication efficiencies and different Ct for the same set of dilutions but I can't work out why? Any help would be appreciated!!

Attached Thumbnails

  • Melt Curve all.jpg
  • Amplification Plot all linear.jpg

Attached Files






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