Contamination? - (Feb/10/2010 )
We have difficulties understanding the cause of no amplification of the target sequence while the endogenous control is well amplified during Real-Time PCR.
The endogenous control (HGPRT) is well amplified in all the wells on the plate, but the target sequence CysLTR1 or CysLTR2 is not amplified in parallel (only in few wells). Both reactions (endogenous control and target) take place in the the same well, only primers are different.
We have increased the amount of initial cDNA three times and still the same results. We have also tried to perform the process with different cDNA sample (from other project) and it works fine, control and target are both amplified.
Could anybody suggest how could this problem be solved? Could contamination affect only the amplification of the target sequence (CysLTR1 or CysLTR2) ?
Sounds like you r doing multiplex PCR.
Have you check is the primer design is optimum for multiplex pcr? and have you run the "singleplex" pcr for each primer sets to optimize their optimum pcr profile?