Bad slopes in TaqMan reactions - (Sep/03/2006 )
I have been trying to set up a real-time PCR to quantitate viral load in the Rotor-gene 3000, but usually the slopes I got were above -3.0 (-2.4 to -2.7, at best).
I am using the ABI TaqMan Universal Mastermix, primers and probes described in the literature, and a commercial DNA sample as a control to produce the standard curve. The curves have good R and R^2 values (above 0.98), but nothing seems to change the slope! I have already tried to change the cycling parameters, to tritiate primers and probe concenrations, and even to add more Mg to the mix. Nothing works. I would be very grateful to receive any comments and tips!
Thank you very much in advance!
Hi!
I have the same problems with the slope, but I am working with cDNA, synthesized from RNA extracted from few cells. Do you know what is quantity of sample you work with?
I am using the ABI TaqMan Universal Mastermix, primers and probes described in the literature, and a commercial DNA sample as a control to produce the standard curve. The curves have good R and R^2 values (above 0.98), but nothing seems to change the slope! I have already tried to change the cycling parameters, to tritiate primers and probe concenrations, and even to add more Mg to the mix. Nothing works. I would be very grateful to receive any comments and tips!
Thank you very much in advance!
What is your target for optimization? Virus or amplimer-cloned plasmids? What do the amplification plots look like, is there log amplification with the TaqMan probe? Have you tried varying the ratio and concentration of probe to primers? I would be surprised if the only problem is the slope calculation, if the amplification plots (i.e. curves) look abnormal, I would re-evaluate the probe concentration and sequence. Is there ROX in your ABI Universal Mix? If so, your TaqMan chemistry cannot conflict with the ROX fluorescence (such as a Texas Red labeled probe). My first study would be to compare PCR efficiencies of the same primer set using SYBR green to determine if it is the probe, or the primers, or a combination of both when the probe is added to the mix, good luck...
C
Dear Elgui,
Did you know the concentration of your commecial DNA sample?
Slope represent your PCR efficiency, and standard curve with slope -2.4 to -2.7 is not good.
You need to check ceveral things:
- primer no degradation
- probe (TaqMan) no degradation
- titrate you standard DNA freshly from a high concentration stock every time before you start.
- no DNA is you tube and water.
I also suggest, you may use SYBR Green as substitution. If SYBR Green give you a good standard curve, slope -3.3, meaning that you TaqMan probe might be degraded.......
Also please attach you amplification curve as well as you standard curve.
Best regards