How to dilute and store SYBR Green I mother liquor? Repeated freeze-thaw cycles will result in failure

There is no fixed value for how much the fluorescence intensity decreases after four to five freeze-thaw cycles; it depends on the volume per tube, exposure time, light exposure, and tube material. In general, more than 2-3 cycles is not recommended. In our comparison, when aliquoted into small tubes to avoid repeated freeze-thaw cycles, the signal remained basically stable over one month of continuous use; however, when the entire bottle underwent more than four freeze-thaw cycles, Ct values for low-copy samples tended to be higher and reproducibility worsened. If you have already undergone four to five freeze-thaw cycles, it is recommended not to use it directly for critical quantification. First run a standard curve and positive control to check whether the amplification efficiency and NTC peak onset are still normal.

The 10,000× stock solution is most vulnerable to repeated freeze-thaw cycles of the entire bottle and exposure to light. We typically aliquot it immediately upon receipt using low-binding, light-protected 0.2 or 0.5 ml PCR tubes, 5-10 µl per tube, just enough for one qPCR reaction, with the lot number and date clearly labeled. During aliquoting, work quickly under light-protected conditions. After aliquoting, wrap the tubes in aluminum foil bags and store them on a fixed shelf at -20°C, away from the door or areas that are frequently opened and closed. If the refrigerator door is opened frequently, it is recommended to place desiccant in the storage box to prevent condensation from entering. If a -80°C freezer is available in the lab, long-term storage is better, but be aware of the freeze-thaw characteristics of DMSO-based solvents.

Do not store 10000 x of working solution diluted directly with water to a very low concentration for a long time, as it is prone to instability. The manual generally recommends fresh dilution with DMSO or a matching diluent, and water dilution is only suitable for short-term use. If performing qPCR, we usually do not dilute separately, but directly add the mother liquor to the master mix for each reaction and mix well to reduce dilution errors. When quantifying plant genomic DNA, it is recommended to directly use the commercial SYBR Green qPCR mix. The manufacturer has optimized the dye and buffer ratio. If you insist on matching it yourself, first verify the template gradient in small batches.

The quantification of plant genomic DNA is sensitive to inter batch differences. In addition to dye freezing and thawing, attention should also be paid to extraction batches, RNA residues, and DNA integrity. We will place the same genomic DNA standard in each plate as a gradient and monitor inter batch offset using quality control samples with known Ct. After dividing the dye into single use tubes, use the same batch number each time and try to prepare a large volume master mix at once before dividing it into each well. If replacing the packaging tube with a new one, it is necessary to run 3-4 gradient points parallel to the old batch number to avoid Ct drift affecting the quantitative results.