Do I have to prepare and add the inducer immediately when using IPTG for low-temperature induction?

After using the same bottle of IPTG for six months, the target protein may decrease, and both storage and strain aging may occur. However, it is more common for the effective concentration to decrease due to repeated freezing and thawing or contamination of the mother liquor, or for the transformation efficiency of the strain/plasmid to decrease or for the target gene to mutate after -80 freezing. Suggest conducting a cross experiment: open a new bottle of IPTG mixed with mother liquor and induce the same batch of fresh transformed bacteria simultaneously with the old bottle; If the expression is restored, it basically points to the inducer; If it is still low, repeat plasmid sequencing or re transform. In terms of strain aging, it is best to use the same batch of competent or fresh streaked monoclonal antibodies to reduce the number of passages. Membrane protein induction itself has low fault tolerance, and it is recommended to regularly update the seeds.

Preparing it fresh during the day and keeping it in an ice box for half a day is usually fine, but repeated use from the "same bottle" of stock solution over six months may be a problem. IPTG stock solution is best prepared at 1 M, aliquoted at 0.5 mL/tube, and stored at -20, using one tube at a time. If your habit is to thaw the entire bottle, take what you need, and freeze the rest again, the titer may decline after six months. It is recommended to switch to aliquoting and label the date. Whether the inducer is prepared fresh and added immediately is less important than stock solution storage and the state of the bacterial strain. When we induce membrane proteins, after adding IPTG, we incubate at 16°C for 20 hours, and the OD should be as consistent as possible with the OD before induction. Your concentration of 0.2 mM is not high, so you may consider retrying with fresh stock solution; if expression is still low, you need to troubleshoot the expression conditions and the plasmid.

If you are concerned, you can do a small verification: divide the same bottle of stock solution into two tubes, place one tube in an ice box for half a day, and thaw the other freshly, then induce the same batch of bacteria separately and check the target band of the membrane fraction. Usually the difference is not obvious. A working concentration of 0.2 mM is relatively low, and if the titer of the stock solution decreases, it may appear more sensitive. Therefore, it is recommended to aliquot into single-use amounts, use one tube each time, and avoid repeated freeze-thaw cycles. If expression levels decrease after half a year, first check whether the stock solution has been repeatedly freeze-thawed or contaminated. In addition, long-term passaging of strains and plasmid loss can also cause decreased expression, so it is recommended to newly transform a batch and compare them at the same time.

It's not necessary to add it now. The 1M mother liquor generally does not degrade to the extent that it affects the working concentration of 0.2 mM after being stored in an ice box for half a day. We perform low-temperature induction of membrane proteins, often thawing the mother liquor on ice in the morning and adding it in the evening, but there is no significant difference in expression levels. The key is that the mother liquor itself needs to be filtered and sterilized, packaged and frozen to avoid repeated freezing and thawing. If the mother liquor prepared during the day has just been freshly prepared from powder, it is recommended to dissolve it completely and filter it before placing it on ice. Do not leave it at room temperature for a long time. OD, temperature, and induction duration have a greater impact on membrane protein induction conditions than half a day of exposure to the inducer.