Problems with amplification curve - (May/27/2008 )
Hi
I'm not happy with the way my amplification curve looks. I get a first plateau after already 1 cycle, and then another increase around 25/35 cycles.
I'm using a pretty standard protocol, where I'm running samples from rat brain that have been snap frozen on dry ice. RNA was extracted with Rneasy kit from Quiagen, and converted into cDNA with the superscript III first stand synthesis system for RT-PCR from Invitrogen. For detection i'm using sybr green PCR master mix from applied bioscience, and I'm using GAPDH as endogenous control.
The melting curve looks fine, and I have also run a gel to make sure that I only have a single product.
I have tried loading 100ng cDNA and 500 ng, running the reaction with 10ul and 25ul reaction volume, but nothing seems to matter.
Can somebody help with what the problem might be????
I would like to try decrease the amount of input cDNA template in this situation. Good luck.
Totally agree. You must have tons of DNA to start seeing peak from the beginning. Dilute your cDNA to 10, 100, 1000x, run a sample reaction, adn choose a dilution where Gapdh starts linear amplification at about 12 to 25 (arbitrarily).
I'm not happy with the way my amplification curve looks. I get a first plateau after already 1 cycle, and then another increase around 25/35 cycles.
I'm using a pretty standard protocol, where I'm running samples from rat brain that have been snap frozen on dry ice. RNA was extracted with Rneasy kit from Quiagen, and converted into cDNA with the superscript III first stand synthesis system for RT-PCR from Invitrogen. For detection i'm using sybr green PCR master mix from applied bioscience, and I'm using GAPDH as endogenous control.
The melting curve looks fine, and I have also run a gel to make sure that I only have a single product.
I have tried loading 100ng cDNA and 500 ng, running the reaction with 10ul and 25ul reaction volume, but nothing seems to matter.
Can somebody help with what the problem might be????
Are you sure that's amplification and not background? How do you set your baseline?
Hi!
Maybe my answer comes a bit late, but what you see at the beginning is not amplification. Some reaction shows this kind of curves probable for the primers)
Do two things:
If your machine can remove blank, do it! (make a control without template and this is your blank)
I would increase the cDNA not decrease, because with none of your genes you are reaching a plateau.
Good luck
It simply looks like your analysis software has applied no baseline correction. Change the settings for baseline correction in the software and this should remove the earlier changes in fluorescence and give you improved contrast for the rest of your plots.