In qPCR, we normally use 45 to 50 cycles of for getting good amplification curve. However, I have some primers which start to get amplification raising peak after 50 cycles and still not complete the amplification within 70 cycles. What should I do for getting good amplification of those primers?
More cycles gives more artefacts. CTs above ~40-45 cycles are unreliable. Optimum amplification should be in the ~15-35 cycle range. To get this, it seems like you need to optimize your template preparation and primer design.
R.K. Kumawat my control genes have CTs of ~15 (highly expressed) and ~38 (lowly expressed). That is the range. Target genes should fall within this range for reproducibility and thus reliability.
Ujjal Kumar Nath another thing, your amplification product should only be ~150bp. You should design a couple of upstream and a couple of downstream primers and test all combinations in a regular PCR to see which pair works best.
Randolp B Cladwell thanks for your suggestions, I will optimize my primers. I designed primers that all are 115 to 135 bp product size.
This depends on your amount / conc. of the target (template DNA).
The more template you have when starting the PCR the less cycles are needed.
If increase cycles 40 to 45 in qPCR florescence will be due to artifact most shulatble cycle in q PCR -30 to 35.
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