One MGB probes works, but the other does not. - (Jun/12/2007 )
I am using a real-time MGB Taqman probes to detect a single point mutation. Using plasmids I can get amplification with the sensitive FAM labelled probe, however when I use the resistant (Vic) probe and the resistant plasmid I do not get amplification but I do get an increase in fluorescence in the component plot. I am using the same primers in each reaction the only thing that is different is the probe and the plasmids. I have checked the plasmid is the correct sequence and this is all fine. Therefore, I am at a loss to understand why the VIC labelled probe does not produce any amplification. At present I will be using the probes in separate reactions with view to multiplexing later. Can anybody help!!!!!
-CucumberPM-
QUOTE (CucumberPM @ Jun 12 2007, 11:59 AM)
I am using a real-time MGB Taqman probes to detect a single point mutation. Using plasmids I can get amplification with the sensitive FAM labelled probe, however when I use the resistant (Vic) probe and the resistant plasmid I do not get amplification but I do get an increase in fluorescence in the component plot. I am using the same primers in each reaction the only thing that is different is the probe and the plasmids. I have checked the plasmid is the correct sequence and this is all fine. Therefore, I am at a loss to understand why the VIC labelled probe does not produce any amplification. At present I will be using the probes in separate reactions with view to multiplexing later. Can anybody help!!!!!
Hi!
When you say you get no amplification with the VIC probe, is it a complete deadline or is there a small rise in fluorescence? If you are getting a signal with the FAM probe from both plasmid types, this does suggest that the sequence is right. I was getting the same problem and found that by changing my reaction mixture from Taqman Universal to Taqman genotyping mix made a really big difference. Hope this helps.
Best wishes
Stand Free
-standfree1983-