iQ5 dynamic well factors for TaqMan probes - (Feb/27/2008 )
I was told by BioRad tech support that I do not need a passive reference dye (such as fluorescein) if I am using labeled TaqMan-type probes. According to them, as long as each well has the same amount of each fluorophore, dynamic well factors can be set collected using the probes themselves. In my case, I will be doing relative quantitation, normalizing to a couple of housekeeping genes. I will use the same fluorophores on all three probes (GOI plus 2 reference genes), but will run all three on one plate. I'm assuming that if the probes are approximately the same length, then if the molarity in each reaction is the same, this should be ok.
So, my question is why then do master mixes such as Qiagen's QuantiTect Probe PCR mix contain ROX for normalization? Is this in case you want to use different fluorophores or what?
Thanks for any input!
As far as I know it is just for the machines that needs the ROX normalization. Many kits I have seen exist in the 2 versions, and the one I am using (Finnzyme dynamo probe qPCR kit) supplies the ROX separately. But the Bio-Rad thermocycler do not need the ROX.
And as far as I know, the probe length should not matter, only the molarity because each probe is labelled only once.
That makes sense! Thank you for your reply.
it is not only the molarity that counts
if you are using the internal / dynamic well factor with 2 different probes you may get problems because each probe has a different degree of quenching so that 2 probes although being at the same concentration have different fluorescent backgrounds which disturbs dynamic well factor calculation...
in the end I always use the external well factor procedure and i am really anoyed of this dynamic well factor procedure it cost me so much nerves and experim. repetitions...
I want my good old light cycler 2.0 back or better a cool lc 480!!!! does anyone want to change one against a iq5????
To be honest, I did not get the explanation from Biorad.
Could anyone please point to some reading, where I could learn more about internal and external dynamic factors?
Would you be willing to tell a bit more about your bad experiences with ROX? Thanks in any case.
Here is the link for the iQ5 manual where they describe the dynamic and external well factor procedure:
Hope this helps you in theory but for a real feeling you unfortunately have to work with the iQ5 instrument...
Regarding Rox and....
I just do not understand why this is all necessary: well factors, ROX dyes, different ROX conc., the real-time PCR world would be so much easier if there were only "high-quality" instruments on the market which can deal with and analyze "raw-data" in a reasonable manner...