I would like to inquire about the possibility of pre-preparing a gene expression experiment. Can I prepare all reaction components (cDNA, primers, and master mix) in the laboratory plate three days prior to the actual experiment date and store them frozen at -18°C?
Hello Sarah,
1. Master mix and primers
Yes, you can. Commercial qPCR or RT-qPCR master mixes are typically stable when stored frozen. You can prepare the master mix with primers in advance, aliquot it into PCR plates or tubes, and freeze it at −20 °C. Many labs perform this step to minimise pipetting errors and expedite their runs. Thaw once, mix, and use.
2. cDNA
No, you cannot. cDNA is stable at −20 °C if stored separately. But once mixed with master mix and primers, the degradation risk increases. Freeze-thaw cycles, nuclease contamination, or primer degradation can affect results. It is strongly recommended to store cDNA stocks separately and add them fresh on the day of the experiment.
Best,
It is best not to pre-assemble qPCR reactions and freeze them, as enzyme activity and assay performance can decline; instead, store cDNA, primers, and master mix separately and combine them fresh before use. If unavoidable, some studies show assembled plates can be stored at −20 °C for a few days if sealed tightly, but you should validate this carefully for your assay.
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