Experiment with DNA and learn the techniques and concepts in a convenient and easy way

[Matías Gutiérrez]

Hello Everyone, 

       I'm rather new to this list and I'd like to introduce myself. I'm a biochemist based in Chile and for about 3 years I've been risking it all in an ambitious business endeavour which seeks to develop inexpensive high quality lab equipment for labs that can't afford the absurd prices companies charge now a days. We've also focused in developing science education products for schools and universities. We've already developed an inexpensive gel documentation platform that broadcasts the pictures wirelessly to your computer and I'm currently working with Guido Nuñez and Joseph Jackson on the development of the portable and inexpensive PCR machine LavaAmp from which you may have heard in the past. They introduced me to this list a while ago and I've been paying close attention to the conversation and I'm personally thrilled by the great momentum this movement is gaining. 

      Among some of the recent topics that interested me was how many of you are interested in an informal yet practical way of learning some of the basic concepts of Molecular Biology, these being PCR, restriction enzime digest, ligation, transformation of E coli or other organisms, etc... For about 4 years I've been developing educational kits for this very purpose, help educators teach these to highschool and college students. Our kits usually contain enough material for 40 ppl to learn through experimentation, yet I've been toying around with the idea of making a simpler version for single individuals for a while and I think this may be the right audience for such a product. 

 

      Currently with our kits you can experiment the following

1. DNA extraction from fruits: This is a very straight forward experiment that was recently discussed, we've optomized reagents and protocols so that anyone can get a great result. The end result is a beautiful DNA tangle that is left suspended (insoluble) in a conical tube. 

2. Transformation of Bacterial cells with the Green Fluorescent Protein gene (GFP): The format that we've developed allows anyone to introduce a plasmid containing GFP gene and ampicilling resistance. Concepts to be applied include heat shock based transformation, selection of transformants with ampicillin, efficiency of transformation and arabinose mediated induction of recombinant protein expression. 

3. DNA fingerprinting using restriction enzymes: Find out "who committed a crime" by comparing the restriction digest profile of the DNA found at the crime scene with the one from 4 different suspects.

Future kits: 

-PCR

-Cloning (insertion of DNA into a plasmid, transformation and selection of transformants)

-Something you suggest

 I would really like to know if you are interested in these, every kit comes with all reagents and consumables required, protocols, a slide show and literature explaining all the concepts involved in the experiment and we also offer support through e-mail. We try to develop the experiments so that you don't need any lab equipment to perform, but the fingerprinting for example requires horizontal gel electrophoresis. 

We currently don't have any distributors outside of Chile, perhaps the Biocurious Reagent Store could serve this purpose for US based users or someone else.

Matías Gutiérrez

Bioquimica.cl 

matias.g...@bioquimica.cl

www.bioquimica.cl 

Cell +569 8228 8405

[C.R.S.]

Dear Matias,

I am a retired (Baylor Coll. of Med. 1992) clinical chemist who now has become interested in learning and teaching what I have learned about micro-propagation and molecular biological
techniques before I slip further into senescence. I wish you good and rapid success with your endeavours and look forward to being able to order some of your equipment.  Deseo que usted continuara y éxito rápido. Edwin S. Katz, Houston Texas

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[Mega]

As for 2. GFP I want to mention, you need arabinose.

You can get it as DIY biologist, but not from the supermarket ( I
think so, in fact never looked for arab there)


And you need a black light source.


There's a plasmid, pVIB, that is always 'on' and needs no special
substrate. It may be more attracting to people if they see it glowing
in the dark without UV-Lamps.
That may only be true for me, at all. Though, I recommend thinking
about this.


Best

[Derek]

Nope, no arabinose at the supermarket. It's found in low
concentrations in grape and apple juice, but I wasn't able to get
sufficient concentrations to enable GFP production.

Best price I found, and nice people to deal with, are the folks at
Cascade Biochemicals, http://www.cascadebiochems.com/

--Derek

[Mega]

So you are still dependent on chemical companies that may deny
access.

And if you (vacuum) destill the apple juice so the concentration gets
higher? Is arab heat stable (say 100°C)?

On 8 Mrz., 20:20, Derek <dere...@gmail.com> wrote:
> Nope, no arabinose at the supermarket. It's found in low
> concentrations in grape and apple juice, but I wasn't able to get
> sufficient concentrations to enable GFP production.
>
> Best price I found, and nice people to deal with, are the folks at

> Cascade Biochemicals,http://www.cascadebiochems.com/

[Avery louie]

You might just want to get pGREEN from carolina.  There is no need for arabinose with that plasmid.

--Avery

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[Matías Gutiérrez]

Hey Guys, 

   First of all thanks to Edwin for your warm wishes :) 

Now regarding these kits, they basically include everything. 

For example the GFP cloning kit includes the following:

• plasmid encoding GFP and ampicilling resistance (GFP expression is induced by arabinose)
• bacterial stock culture (stab) that needs to be propagated before the experiment
• 2 plates containing propagation media (LB)
• plate containing selection media (LB + ampicillin)
• plate containing selection media and inducer (LB + ampicilling + arabinose)
• inoculations loops, microtubes, transfer pipettes, etc
• detailed protocol for the experiment
• Slide show with explanation of the protocol
• further reading material 

If we can work out a good distribuition network I'd really like to make this available for the community at a low cost (hopefully less than USD$30.00)

Matías

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[Nathan McCorkle]

Matias, what about liquid LB for propagation?

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--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

[Matías Gutiérrez]

Also included, forgot to add it into the list. :) but not for propagation, the propagation is achieved by plating the bacteria in the solid LB media. The liquid LB is used for recovery after heat shock transformation. 

Basically the idea is that you'll have everything you need, we've tested these in classrooms for over 3 years with great results and have improved our kit each year, this would be the first version that is not meant for the classroom but for enthusiasts! 

We could also provide individual components in a convenient ammount so that ppl don't need to buy bulk like research labs usually do. 

Matías

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[Nathan McCorkle]

Does your kit require a centrifuge for the CaCl2 rinses? Or have you
got around that with a tweaked protocol?

On Thu, Mar 8, 2012 at 7:20 PM, Matías Gutiérrez

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>> Nathan McCorkle
>> Rochester Institute of Technology
>> College of Science, Biotechnology/Bioinformatics
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[Matías Gutiérrez]

Sorry about the multiple e-mails, but I also forgot to mention that it includes a UV led lamp so that you can visualize fluorescence, it looks really bright, like this:

[Matías Gutiérrez]

Nathan, 

       You literally need no equipment for the GFP cloning kit. If you have a lab you will use a water bath for the heat shock at 42°C and a 37°C dry incubator to grow the bacteria in the plates provided. Yet if you don't have these you can just mix hot and cold water until a thermometer tells you you've reached 42°C just before the heat shock in the transformation step, and as far as incubation goes there are two options we recommend, the easiest is tu place the plates behind your fridge where it gets warm and if you want to be more sophisticated you can just put a 40W old-school light bulb into a cardboard box the size of 4 six-packs and you'll reach a temperature close to 37°C (test with a termometer). 

Matías

[Heather]

I think the kits sound amazing! One I would be interested in for my
own purposes is a genetic analysis kit that would allow comparison of
human dna for various traits. I would be happy to discuss in more
detail off-list if you like.

Heather

On Mar 8, 6:46 pm, Matías Gutiérrez <matiasgutierr...@gmail.com>
wrote:

> Hey Guys,
>
>    First of all thanks to Edwin for your warm wishes :)
>
> Now regarding these kits, they basically include everything.
>
> For example the GFP cloning kit includes the following:
>

>    - plasmid encoding GFP and ampicilling resistance (GFP expression is
>    induced by arabinose)
>    - bacterial stock culture (stab) that needs to be propagated before the
>    experiment
>    - 2 plates containing propagation media (LB)
>    - plate containing selection media (LB + ampicillin)
>    - plate containing selection media and inducer (LB + ampicilling +
>    arabinose)
>    - inoculations loops, microtubes, transfer pipettes, etc
>    - detailed protocol for the experiment
>    - Slide show with explanation of the protocol
>    - further reading material

>
> If we can work out a good distribuition network I'd really like to make
> this available for the community at a low cost (hopefully less than
> USD$30.00)
>
> Matías
>
>
>
>
>
>
>
> On Thursday, March 8, 2012 6:29:05 PM UTC-3, Avery wrote:
>
> > You might just want to get pGREEN from carolina.  There is no need for
> > arabinose with that plasmid.
>
> > --Avery
>

> >> diybio+un...@googlegroups.com.

[Dakota Hamill]

Your website looks great.  I like all the pictures and the whole thing has a sleek, polished, futuristic look. 

 For me, I'd be more interested in those nice poly-acrylic little PCR workstations you have, as well as the Neon labware products.  The GelDoc looks like a fantastic piece of hardware, although probably out of my price range.  I just helped install a UV gel imager donated to my university yesterday and it was about half the size of a refrigerator (so 4-6x the size of the gel doc) and had about 12 wires coming out of the thing.  So, a compact wireless alternative seems like it could fill a nice market niche.  

As for the kits, they look great, but there is already competition for those same exact kits in the US, ie, crime scene digest, pGREEN kit etc.  I am not saying they wouldn't sell if you did end up getting a place to carry them in the US, just already big places like Carolina  that do them as well.  As an individual who has already done them or things like them in High School/University, I most likely wouldn't be interested in them as an individual (not trying to sound rude, just truthful), but I think they fit perfect for the target audience of middle/high school or DIYBio individuals who haven't done them before.    I am more interested in cheaper access to reagents and "DNA parts" then fully assembled kits, but I'm just one out of 2,000+ on this list, so take everything I say with a grain of salt!

Although, it would be fun to teach a public course at a local high school on the weekends or something about bio-tech, and a fully assembled kit made for 40 people would be a great thing to just buy ready, and not have to assemble yourself.  

So in conclusion, that sleek looking GelDoc, PCR Workstation, Pipette rack, gel box are what look pretty sweet to me.

Best of luck! Hope to see you around on the list

-Dakota

[Nathan McCorkle]

Can you post the protocol? I know there have been people on the list
lately that have been trying to manage a GFP transformation without a
centrifuge (you didn't address how you get around this).

On Thu, Mar 8, 2012 at 7:20 PM, Matías Gutiérrez

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>> Rochester Institute of Technology
>> College of Science, Biotechnology/Bioinformatics
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[Matías Gutiérrez]

Nathan, 

        As far as I know, the purpose of CaCl2 is no neutralize the negative charge of the DNA and on the bacterial membrane so that the plasmid is not repelled by the membrane. What we do is take a colony off a plate and then transfer them into two tubes, one containing CaCl2 and the plasmid and a control tube containing only CaCl2. Bacteria are resuspended using a loop and that's all you need before going into heat shock protocol. 

        For this particular experiment, we use a large amount of DNA to achieve transformation. Competent cells are obtained from fresh colonies of E coli growing in solid LB media, they are resuspended in CaCl2 so there is no need to wash LB off, since you are using solid media to propagate. So basically the transformation efficiency is not great and we compensate for that by using a lot of plasmid DNA to get the result. In our lab, when we want to clone something we've made by cutting and pasting DNA we need to prepare competent cells. Since we don't have a large centrifuge for bulk competent cell peparation - what we do is grow E coli untiil log phase then transfer 1.5 mL of culture to 1,5 microtubes, spin in a centrifuge at 6K RPM, add more culture into the same tube and repeat, and then resuspend in CaCl2, these are ready and competent and can be frozen at -20°C for later use, though I don't think they remain competent very long. 

Matías

A 

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[Mega]

What I'm interested in:


- There is a safe culture of E.Coli (K12 or something) in the kit?
- You need a centrifuge?
- What are shipment costs? I assume that the plasmid is stable also
at room temperature for some time. So there would be no parts that
have to be frozen on arrival, which makes transport costs smaller.

[Matías Gutiérrez]

Hey Mega, I'll reply in text below

On Friday, March 9, 2012 11:45:16 AM UTC-3, Mega wrote:

What I'm interested in:


- There is a safe culture of E.Coli (K12 or something) in the kit?

We ship with DH5a which is harmless 

- You need a centrifuge?

No, 

- What are shipment costs?  I assume that the plasmid is stable also
at room temperature for some time. So there would be no parts that
have to be frozen on arrival, which makes transport costs smaller.

 

Shipment does not require special handling, items should be stored at 4°C (fridge) upon arrival, the only labile items are the media plates.  Shipping cost will depend on who will be distributing. Where are you located?

Matías

[Mega]

I'm in Austria (Europe)
Shipment in my country is always a big issue.

For example: pVIB was 9,10$. That's ok. But shipment was 35$! (I
bought 2 quantities -> shipment per tube: 12,5$. )

One time this is Ok. But if you do this often, your budget is consumed
just for transportation.




I'd definitely be interessted in a GFP kit.


On 10 Mrz., 17:46, Matías Gutiérrez <matiasgutierr...@gmail.com>
wrote: