Re: [DIYbio] Re: Is there interest in lower cost restriction enzyme varieties?

https://www.neb.com/products/e0546-biobrick-assembly-kit is avalioable
for biobrick assembly.
Not exactly what you are wanting but interesting, if a little pricey still.

The BioBrick® Assembly Kit provides a streamlined method for assembly
of BioBrick parts into multi-component genetic systems. BioBrick parts
are DNA sequences that encode a defined biological function and can be
readily assembled with any other BioBrick part. The process for
assembling any two BioBrick parts is identical and results in a new
composite BioBrick part.

The BioBrick Assembly Kit contains EcoRI-HF™, XbaI, SpeI, PstI, T4 DNA
Ligase and NEBuffer 2.1.

On Sat, Nov 15, 2014 at 6:25 AM, Koeng <koeng101@gmail.com> wrote:
> 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:
>>
>> 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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Brian Degger
twitter: @drbrian

http://makerspace.org.uk
http://transitlab.org
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