This makes sense. Some enzymes are more resistant than others though. BbsI, for example, begins to degrade at -20.
-- I would like to know if there is a relationship between protein stability at room temperature and heat inactivation... I'd assume so since its all about protein stability. BamHI can't be heat inactivated (super unfortunate for BglBrick assembly, however I am actually currently discussing with NEB about one of their variants that is heat inactivatable! Gotta have BglII too though).
It seems like another simple experiment could be in place here. Keep a stock of diluted BamHI at room temperature and a stock of diluted BamHI at -20. (dilution because enzymes are expensive man). Keep these both at their temperatures for a month. After that month, take them both out and do serial dilutions and run it on some lambda DNA, then do a gel. The dilutions will eventually be below a unit, and then not cut all of the lambda DNA. In a perfect world, this would be at the same dilutions of both. In a realistic world, I would estimate a ~75% loss of activity because I am a pessimist.
If I get time next week I'll set up that experiment
btw, sorry for changing the topic on the thread. Really if you want good enzymes, just work to buy em from NEB. I talked to one of the price specialists a week or 2 ago and the main reason they do the quantity is because they want a universal base price of 'around 50 dollars for 500 units' universally. Most labs only use a few enzymes anyway, and a variety pack wouldn't really be useful to *most* labs because there isn't a need. They already have their cloning systems down and know what to use. If enough DIYbiologists get together to actually ask, however, and show there is a demand for it, I don't see any reason why they wouldn't go for it, other than it is hard to do that little of enzyme in a tube. If it comes to expressing it yourself, I have SpeI and XbaI fully synthesized with their methylase, you just need to actually clone it into an expression vector. Purification is a whole other issue, one I'm not skilled at :) . I honestly think it is hard to beat NEBs prices
-Koeng
On Friday, November 14, 2014 11:24:03 AM UTC-8, Mega [Andreas Stuermer] wrote:
On Friday, November 14, 2014 11:24:03 AM UTC-8, Mega [Andreas Stuermer] wrote:
There is that paper that says some restriction enzymes are stable at room temperature for 6 months - and still retain like 99% activity after that period IIRC.I don't remember which enzymes it were (BamHI?)... The paper was discussed here a year ago or so...If you kept them at 4°C or -20°C that would surely be better. And available.
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