How many times can dNTPs be repeatedly frozen and thawed when stored at -20 ℃?

The shelf life of dNTPs stored at -20°C is typically 12-24 months, but this refers to unopened original products or those stored as recommended. After aliquoting, use them within the original shelf life as much as possible, and be careful not to exceed the expiration date. If pre-aliquoted products are for single use, use them immediately after opening, and there is no concern about repeated freeze-thaw cycles. The 10 μL/tube or 20 μL/tube packaging you asked about is suitable for taking one tube at a time, avoiding refreezing the remainder. During storage, place them toward the back of the refrigerator where temperature fluctuations are minimal, not near the door. If pH changes, color darkening, or abnormal amplification occurs, stop using them. It is recommended to record the production date upon new arrival, follow first-in-first-out, and regularly discard expired reagents. If your experiments are sensitive to contamination, you can take one tube before using aliquoted or pre-aliquoted products to perform a no-template control validation.

DNTPs are stored at -20 ℃, and it is generally recommended to minimize repeated freeze-thaw cycles. The common saying is not to exceed 5-10 times, but this is not a hard standard. High quality products may tolerate more cycles. Repeated freeze-thaw cycles can accelerate micro hydrolysis, especially dGTP and dATP, which may oxidize or degrade, leading to a decrease in PCR amplification efficiency or non-specific results. You use a small amount. If you don't pack the 100 mM large package separately, the risk of repeated freezing and thawing of the entire bottle is relatively high. I usually immediately pack the goods on ice into single use quantities, such as 10 μ L or 20 μ L per tube, and discard each tube once to avoid repetition. After packaging, mark the date and batch number, and store in the dark at -20 ℃. Also, when using, melt on ice and do not let it sit at room temperature for too long.

Aliquoting it yourself can indeed introduce nucleases or microorganisms, but proper technique can greatly reduce the risk. I will work in a clean bench or PCR workstation, use filter tips and nuclease-free low-binding tubes, wear clean gloves and change them frequently. Before aliquoting, wipe the bench surface with 70% ethanol, briefly centrifuge the original dNTP tube after removing it, and place it on an ice block to avoid warming it with hand heat. The aliquot volume must be accurate, and repeated pipetting should be avoided to prevent bubbles. If the laboratory does not have a clean bench, at least choose an area free of PCR product contamination, and do not aliquot in the place where PCR products are handled. After aliquoting is completed, take one tube for a negative PCR test to confirm that there is no contamination. Although pre-aliquoted products are convenient, aliquoting them yourself allows more flexible matching of usage amounts.

There are already many options for domestically produced pre packaged dNTPs, and disposable packaging of 10 μ L/tube or 20 μ L/tube is suitable for small-scale laboratories like yours. When making a purchase, I usually check whether it is DNase/RNase free or nuclease free; Whether to use low adsorption and light avoidance tubes; Is there a CoA indicating purity and concentration; Is the production date and expiration date clear. In terms of price, pre packaged items may be more expensive than large packaged items, ranging from a few yuan to over ten yuan per tube, depending on the brand and specifications. Bulk purchases may offer discounts. Some products are made into freeze-dried or contain protective agents, and you can inquire about the storage conditions. Suggest buying small packages for trial first, running several rounds of regular PCR and qPCR, confirming normal amplification, and then fixing the supplier.