Mg curve for real-time PCR - Strange result (Sep/21/2005 )
hello everyone,
I made an Mg curve test on 2 new primers to check if I was able to loweer Ct values wich was
33-35.
I'm Using Qiagen PCR SYbr Green Kit, and I had prepared several mixes with different Mg concentrations.
When I ran My samples Mg appear to be not affecting Ct values. In the past I Made the same test with other primers and I obtained different Ct values.
Did anyone experienced the former case? Is this normal?
Thanks in advance (Sry for the bad english 
)
Dear BioGothmog,
Ct value is directly determine by the amount of your DNA template, provided primer and MgCl2 concentration is not a limitting factor. What is your [Mg++] please?
In your past experience, your [Mg++] probably is limitting your PCR efficiency, thus when you add more, the Ct value increased.
In order to make your Ct value earlier, I think is more appropriate to add more DNA template.
Adding more Mg will change your PCR product melting behavior rather than Ct value.
Best regards
This is going to vary greatly on the template which you are amplifying. The previous poster is correct that the Ct values are a measurement of the copy number of your target sequence. What you really need to do is perform a standard curve with know concentrations of target and find out the slope of the standard curve. This will tell you the "efficiency" of your PCR reaction. There are many protocols and posts of this website that relate to this procedure.
sry, I think to be misunderstood cause I did not explained well.
I'had learn that Mg (Final concentration of my standard mix= 2,5mM) can affect the Taq Efficency And usually a grater Mg concentration lower the Ct values (I used higher Mg concentration for my tes=3,0-3,5-4,0-4,5-5,0mM) cause the enzyme works more.
But this not happen in my experiment in this case while it happened in my previous tests... I hope to have straightened my question!
then I wanted to know: is this normal?
Reading Hadrian answer I think it is, if there are some other suggestion I here to learn more!!
ANyway thanks for your answers
Bye
!
Dear BioGothmog,
As what you say, it is normal.
If you have some chemical to spere, I think is batter for you to perform a standard curve (10 fold serial dilution) in various Mg concentration, let say 1.5, 2.0, 2.5, 3.0.......4.0mM.
From that you can select the Mg concentration which give you the best PCR efficiency (<95%, slope -3.3).
After you got your optimum Mg concentration, all you need to do is increase the amount of your initial template to make your Ct ealier.
Best regards
Dear BioGothmog,
As what you say, it is normal.
If you have some chemical to spere, I think is batter for you to perform a standard curve (10 fold serial dilution) in various Mg concentration, let say 1.5, 2.0, 2.5, 3.0.......4.0mM.
From that you can select the Mg concentration which give you the best PCR efficiency (<95%, slope -3.3).
After you got your optimum Mg concentration, all you need to do is increase the amount of your initial template to make your Ct ealier.
Best regards
That was what I thougth... now y confirmed this I am more sure and I will work on it.
Thanks for your kindly answer!