Currently I process a commercial Multiplex Realtime PCR kit on Quantstudio 5 system, but almost 20% of the runs has a distorted amplification plot (see pictures attached below).
Does anyone suffer this situation? What can I do to improve it?
Thanh Tran I need more details of your analysis? How much primer did you use? How much pairs of primer and probes did you use?
Giorgia Pertile I use a commercial kit which is optimized by the producer. We do not have any official information about the concentration or number of primer pair/probes. I think that they may be use 2 set of primer pairs and 2 probes corresponding to 2 targets. The target size is about 200-400 bp each. Below is the thermal cycle program.
Thanh Tran Ok. first id you use the Taqman you need to run only one step. For example 95°C for 5 min; 40 cycles of 95°C for 15 sec, 60°C for 4 min (in this step you make the data collection).
When you try to optimise the reaction, you need to run the same concentration of DNA and try to run in simpleplex and multiplex.
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