Can SYBR Green dye still be used with precipitation? Has a significant impact on Ct values

Be cautious with filtration. Ordinary 0.22 um filters may adsorb the dye, causing the actual concentration to decrease. Moreover, if the precipitate is the active component, the signal will be lower after filtration, and concentration calibration becomes troublesome. If this is not a critical experiment, I recommend simply replacing it with a new one. If you insist on using it, first centrifuge at low speed and take the supernatant, then make up to the original volume with a small amount of DMSO or a suitable diluent, but you must rerun the standard curve. Do not directly prepare the premix from a stock solution containing precipitate, to avoid ruining the entire plate. Dyes stored at 4 degrees for a long time, especially those opened for more than 1-2 months, carry higher risk.

Flocculent precipitate at the bottom that disperses when shaken is usually due to localized dye precipitation after prolonged storage at 4°C or temperature fluctuations during transport, and may also involve adsorption by the container material. After brief centrifugation and gentle mixing, if it completely disperses and the liquid is clear, you can first run a small-sample test; however, if fine particles remain after mixing or oily aggregates appear on the liquid surface, it is advisable not to load it directly onto the quantitative plate. SYBR Green I is a concentrated dye, and precipitation makes it difficult to control the local concentration. A 0.5 µL sample volume itself carries considerable error, posing a greater risk to well-to-well reproducibility than one might expect.

This type of precipitation is not uncommon in dyes that have been stored for a long time at 4 degrees Celsius. In the future, it is best to divide SYBR Green I that has been opened into small tubes according to a single usage, and wrap them in brown light blocking tubes or aluminum foil to avoid repeatedly taking them out from 4 degrees and reheating them. When adding samples to a 96 well plate, first equilibrate the dye to room temperature, gently invert and mix well, and then aspirate; It is recommended to use a calibrated pipette with a volume of 0.5ul and pre mix the dye into the master mix instead of adding the dye first to reduce inter pore errors. If the sample is precious, conduct preliminary experiments before making a decision.

It is not recommended to directly determine whether there is any impact. The safest approach is to use the same mouse cDNA or plasmid standard, continuously add 8-16 wells, and compare the Ct mean, SD, and melting curve peak height of this bottle of dye with previous normal batches. If the Ct SD changes from below 0.2 to above 0.5, or the melting peak becomes significantly wider, it indicates uneven dispersion of the dye. You can also let it sit and only take the supernatant to run the standard curve, and compare it with the sample taken after shaking the whole bottle evenly; If the difference in Ct between the two exceeds 0.5, it cannot be used for quantification.