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

I think shipping is a big issue.
I run a DIYBio store, The ODIN (http://www.the-odin.com), and I have purchased and shipped many "thermostable" enzymes and tested them. There is a marked decrease in activity after shipping. Depends what you are trying to do but usually this is not a good thing.

If people know the quality of enzyme they are going to receive is not great then it might work. Most restriction enzymes at NEB and such are not really cost prohibitive these days at ~$50-$100 USD. Especially if you do experiments at a community lab and can find someone to split it with you. I usually stick with two enzymes for all my cloning(NdeI and BamHI) this saves alot of time effort and money.

Of course these days there are also many methods that are restriction enzyme free for cloning.

On Saturday, November 15, 2014 4:43:49 AM UTC-8, DrBrian wrote:

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 <koen...@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
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