slope is not even close -3.22 - (Sep/27/2007 )

Hey everyone

I work with Taq Man probes, two in a mixture, so I can't adjust any of them. So are the primers as well included in the mixture.
Now I want to amplify 2 DNAs in a mixture (each of the DNA is homozygous for the same SNP but opposite alleles). I created a standard curve by mixing two DNAs in known ratios and decreasing concentration of one of the two DNAs - which I expect to be the minor one in the mixture (accordingly the concentration of the 2nd DNA arise) to total 50ng DNA.

Here is my problem... usually the slope should be around -3.22 what means the efficiency of my PCR is 100% (1Ct-difference means double amount of DNA product). So far so good. But my slope when analyzing the curve of the decreasing amount of DNA which is detected by the FAM probe is much too high (-4 to -5). On the opposite the slope is around -2.5 when detecting the alleles specific to VIC probes. Does this value matter anyway when I use a standard curve to get relative amounts? Because these values are reproducible and the given values are close to my input ratio.
Btw I use the TAq Man Genotyping Mastermix and the predesigned TaqMan Genotyping probe mixes. Maybe it has sth. to do with the Mastermix (there are no Mg concentrations given or anything else )

Heeelp...

Why do you need to concern abot the slope....??Since you are doing SNP study.

Best regards

Hey everyone

I work with Taq Man probes, two in a mixture, so I can't adjust any of them. So are the primers as well included in the mixture.
Now I want to amplify 2 DNAs in a mixture (each of the DNA is homozygous for the same SNP but opposite alleles). I created a standard curve by mixing two DNAs in known ratios and decreasing concentration of one of the two DNAs - which I expect to be the minor one in the mixture (accordingly the concentration of the 2nd DNA arise) to total 50ng DNA.

Here is my problem... usually the slope should be around -3.22 what means the efficiency of my PCR is 100% (1Ct-difference means double amount of DNA product). So far so good. But my slope when analyzing the curve of the decreasing amount of DNA which is detected by the FAM probe is much too high (-4 to -5). On the opposite the slope is around -2.5 when detecting the alleles specific to VIC probes. Does this value matter anyway when I use a standard curve to get relative amounts? Because these values are reproducible and the given values are close to my input ratio.
Btw I use the TAq Man Genotyping Mastermix and the predesigned TaqMan Genotyping probe mixes. Maybe it has sth. to do with the Mastermix (there are no Mg concentrations given or anything else )

Heeelp...

Hey everyone

I work with Taq Man probes, two in a mixture, so I can't adjust any of them. So are the primers as well included in the mixture.
Now I want to amplify 2 DNAs in a mixture (each of the DNA is homozygous for the same SNP but opposite alleles). I created a standard curve by mixing two DNAs in known ratios and decreasing concentration of one of the two DNAs - which I expect to be the minor one in the mixture (accordingly the concentration of the 2nd DNA arise) to total 50ng DNA.

Here is my problem... usually the slope should be around -3.22 what means the efficiency of my PCR is 100% (1Ct-difference means double amount of DNA product). So far so good. But my slope when analyzing the curve of the decreasing amount of DNA which is detected by the FAM probe is much too high (-4 to -5). On the opposite the slope is around -2.5 when detecting the alleles specific to VIC probes. Does this value matter anyway when I use a standard curve to get relative amounts? Because these values are reproducible and the given values are close to my input ratio.
Btw I use the TAq Man Genotyping Mastermix and the predesigned TaqMan Genotyping probe mixes. Maybe it has sth. to do with the Mastermix (there are no Mg concentrations given or anything else )

Heeelp...

@ Hadrian

The slope is important or better the problem with the slope is important to me because I am not genotyping - i do a absolute quantification! So I think the slope is important for the result (starting amount of DNA depends on the Ct).
Maybe my explanation what I am doing was a bit confusing.

I use the PREDESIGNED ASSAYs means there are two probes each for one allele and two primers (they come in a ready for use mixture, no adjustment possible). One probe labeled with VIC detects my allele 1 the other probe labeled with FAM detects my allele 2 (each allele comes from different DNA mixed together in an unknown ratio and my job is to find out in which ration they are mixed).
So I tried first simply compare the Cts and calculate the ration (possible if the PCR efficiency is close to 100%) - but that didn't work, because the efficiency wasn't 100% when using a mixture of 2 DNAs (detection of alleles using only one DNA worked fine; great efficiency, great specifity and so on).
Next I tried the standard curve method. I thought if I imitate the mixture of those 2 DNAs (in my unknown sample) in well-defined ratios (like u usually do a standard curve) it must be easy to get the ration in my unknown sample via the standard curve. But here is the problem: If I analyze the standard curves (the one for the allele 1 (VIC) and the second for allele 2 (FAM)) both have different slopes. So I can't use those standard curves for interpreting what I really have in my test tube. And I don't know why the efficiencies are so different when using a mixture.

Thanks for helping me.

I think you actually do a relative quantification if you only need the relative ratio of the two mixed samples. I'd doing somethig similar right now, but not in a single tube reaction.

When talking about standard curves, are they linear or efficiency varies from sample to sample? How does the maximum fluorescence of both probes look like is it similar? (picture?)
If it's stable, you don't need to care for the differences, just count the amounts from the standard curves, using the delta ct method (usually done by absolute quant software) and then divide the ratios to get the mixed sample ratio.

If you want to find the reason why is the efficiency different, that first it's good to examine the SYBR Green I melting profiles of various samples, if there is only single amplicon. If so, then the reason maybe in incomplete binding of one of the probes to the target.
I also encounter assays, where SYBR shows efficiency similar to 2 (100%) and with taqman probe efficiency is lower, not sure why appart from the possibility mentioned above.