Problem with probes? - (Apr/03/2008 )
i had ordered TaqMan probes labeled with FAM and BHQ from AB. They are supposed to come in a lyophilized form at -20, but they had come in hydrated form at 4 degrees.
I am trying to perform my real time pcr, i am getting amplification (seen on agarose gel) but there is no amplification shown in real time. I repeated the expt with diff primer, probe and template conc. but still i m getting the same results.
I tried the reaction with SYBRgreen, but even that is not working, the Ct values i m getting are:
2000ng 9 !!
please help me on this.
by looking at the numbers it seems to me that you are inhibiting the pcr reaction by putting in too much template.
at 200 ng you are getting a nice amplification with a Ct of 19 which doesn't improve by putting in the double amount (400ng) and is even worse with higher amounts -> inhibition.
try much lower amounts of template e.g. 10ng (you should then get a Ct in the mid 20ies).
you can also try a standard curve from e.g. 100ng down to 0.1ng