We had ISO buffer that I made in 2013 in the freezer but another postdoc realized the buffer had turned brown and the Gibson Mix we made with it didn't work. I wondered if others have noticed this problem and if there is a shelf life for ISO buffer used to make Gibson Assembly master mix? We make our own as it generally works better and is cheaper than the commercial mix. My protocol is here: https://benchling.com/protocols/L3HVM4/gibson-assembly . I wondered if anyone knows what caused ISO to turn brown, if they have had similar problems with old ISO buffer, and if there is a reagent that can be left out to prevent this from happening.
If you had several freeze-thaw cycles and kept a solution for a relatively long time at room temperature, the enzymes are unstable.
The assembly mixture can be stored at -20 °C for at least one year.
Yes, I know that Gibson mix itself needs to be kept at -20. I am talking about the ISO buffer component specifically. Generally we'd make a lot of this buffer and then add the enzymes to it when we needed more Gibson mix. We store the ISO buffer at -20. However a postdoc I work with realized that our old buffer turned brownish and the Gibson mixes made with the buffer were not as effective. I wonder if anyone else has had these problems or what the solution is. Perhaps store the ISO buffer aliquots at -80? I am not sure.
Your question is legitimate. Science is dealing with reproducible data. So far you have only one experimental observation.
I had some ISO buffer that started to turn orange after 3-4 years. I think we had some issues with cloning efficiency at the time but I can't say for sure that it was the ISO mix. The color change may be due to PEG degradation. See this reference: https://pubs.acs.org/doi/full/10.1021/ie9015792?src=recsys
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