How long can restriction endonucleases be stored stably at -20 ℃ after being packaged in large packages?

In terms of cost-effectiveness, large packaging should be compared by unit price, and NEB, Thermo, and Takara's large packaging can all be inquired through agents; The import lead time may be 1-2 weeks, while the domestic nearshore/Novartis lead time may be shorter. Don't hoard too much just because the total price is low, as there are still risks associated with long-term storage of restricted enzymes. Our laboratory usually has two people buying together. After packaging, a portion is placed at -20 ℃, and the spare tube is placed at -70 ℃ for stability. However, when using at -70 ℃, repeated freezing and thawing should be avoided.

Repeated freeze-thaw cycles are the main risk, and aliquoting into small tubes is specifically to ensure only one freeze-thaw cycle. After each removal, thaw on ice, and immediately return to -20°C after use; do not leave at room temperature. Storage temperature should preferably be stable, and avoid locations in frost-free freezers where the automatic defrost cycle occurs frequently. If you are concerned about activity after six months, take one tube and perform a restriction digestion titration, for example, 1 U to digest 1 μg of plasmid for 30 min, to see whether digestion is complete; if digestion is incomplete, appropriately increase the amount of enzyme.

When packaging HindIII in large quantities, do not dilute it. Instead, use the original solution and directly package it into sterile low adsorption PCR tubes, with each tube containing one dose or one week's worth. Because the glycerol concentration in the storage buffer of the original solution is stable, once diluted to a low glycerol environment, the enzyme is prone to deactivation. The entire packaging process is carried out on an ice box, with the batch number, date, and U-number marked on the tube wall. When placed at -20 ℃, avoid placing it by the door and use a fixed freezer to store it inside. The activity usually does not significantly decrease within six months.

If activity decreases after aliquoting, first check a few causes: whether the aliquot tubes are sterile and nuclease-free, whether they were left at room temperature too long during aliquoting, whether the glycerol volume has changed due to evaporation, and the number of freeze-thaw cycles. Do not use ordinary water or TE as a diluent for long-term storage; if a working solution is needed, it is recommended to prepare it fresh immediately before use, not overnight. Before buying bulk packaging, you can also ask the distributor to provide aliquoting services or directly buy pre-aliquoted small tubes, eliminating the risks of doing the aliquoting yourself.