Problem with probes? - (Apr/03/2008 )

Hey guys,

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:

template ct
200ng 19
400ng 19
800ng 27
1000ng undetermined
2000ng 9 !!

please help me on this.

-alok

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