Howdy,
On Saturday, February 15, 2014 3:10:12 PM UTC-5, Sebastian wrote:
-- Interesting conversation so far. I've had good results from PCR extension methods, but I think overall they do end up being more pricey than classic ligation.
But, classic ligation works well too. But, to get that consistently successful, you need to keep track of DNA concentrations and purify each digestion.
If you really want to cut down and just clone something, I would suggest checking out Genomikon:
It'll still run you $300-400, but I think you get almost all of the components that you need, minus a bit of equipment. I would double check that they include bacteria though. I've seen this used in beginner cloning workshops twice now, and both times I've been surprised with the robustness. It comes with a bunch of DNA, which will express different colours (green, blue, red), ligase, powdered agar mix, bacteria (I think) and other stuff needed specific for this kit (magnetic beads, magnets, wash solution, etc.)
What you will still need:
Micropipettes (though it might actually work with something like a capillary tube, or pasteur pipette?)
Petri dishes for growing the bacteria on agar (can be replaced by any other container)
The original concept for the kit is explained here: http://2010.igem.org/Team:Alberta
But I think it has since been upgraded. It will come with a protocol though.
It differs a bit from your classic ligation, but learning it will give you all the concepts and more.
I think it's a good place to start. But if you really want to do some engineering, the previous lists/references are great.
For curiosity: Has anyone else used/heard stuff about the Genomikon kit?
Yeah! IndieBB! http://www.indiegogo.com/projects/indiebb-your-first-gmo Start building your personal DNA library today! Woo!
Kevin
@BricobioMTL
On Saturday, February 15, 2014 3:10:12 PM UTC-5, Sebastian wrote:
Personally I like the old school techniques of bacterial amplification
and overnight ligations. I really don't like Gibson for price and error
rate for large bits. Its great for quick work but at $10/rxn Ill pass.
Im in no immediate rush and the wait time gives me extra time to think,
plan, and double check original strategy. In vitro cloning is all fine
and dandy but people spoiled by school teams tend to overlook the
absurd costs involved in using these Ferrari enzymes. I would recommend
shopping around for enzymes and comparing prices including shipping and
asking for a one time new lab discount and but them all in one shot.
NEB offers 50% off of one order. I got all my enzymes in one shot for
half price. If they don't offer discounts remind them that their
competitors do. Negotiation will drastically reduce price. I seldom
ever pay sticker price for anything lab related. Sometimes just asking
for a quote automatically drops prices. Also, get to know your regional
supplier agent. They can hook you up. One last tip is to be extra
aggressive in your discount requests around December since they need to
move all of last years stock off the shelves asap. Asking for samples
is a good start. Ask even if they don't advertise samples. The art of
negotiation has damn near been forgotten. At the end of the day you are
dealing with people, and they can be swayed. Good luck with your lab!
PS if you live in the US and are seriously considering doing biotech as
a hobby/semi-pro, consider forming an LLC to get a business behind your
purchases. They will be more likely to ship if you are not an
individual. Worst case find a local business willing to receive
shipments. A business or nonresidential address is more important and
will open more doors than just an LLC with a res address.
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D From: DIY BIO Groningen
Sent: 2/15/2014 4:53 AM
To: diy...@googlegroups.com
Subject: Re: [DIYbio] Re: Gene splicing equipment?
PCR cloning is the cheapest option most of the time ( you only need to
perform PCRs and in some case blunt self-ligation, Gibson assembly is
great ant time saving in case of multiple gene insertion or other
extensive. Classic restriction enzyme method is very good gym :)
Sent from my iPad
> On 14 Feb 2014, at 22:37, Cathal Garvey <cathal...@cathalgarvey.me> wrote:
>
> Alternatively, gibson assembly, but that's a whole other story. No
> ligases but a one-time cocktail of (3?) enzymes.
>
> I'm making a point of putting nice primer sites on IndieBB if it goes
> ahead so that it'll be gibson-friendly out of the box. :)
>
>
>> On 14/02/14 18:12, W. Estell wrote:
>> They will also need a DNA ligase.
>>
>> On Thursday, February 13, 2014 6:22:38 AM UTC-8, Mega [Andreas Stuermer]
>> wrote:
>>>
>>> I read that some people call editing DNA gene splicing.
>>>
>>> Basically what you need is plasmid DNA (
>>> http://www.indiegogo.com/projects/indiebb-your-first- ) andgmo
>>> restriction endonuclesases. And micropipette 10-100 uL plus 1-10 uL plus
>>> the tips plus tip boxes.
>>>
>>> And DNA you want to put into, and has the same restriction sites.
>
> --
> Please help support my crowdfunding campaign, IndieBB: Currently at
> 23.1% of funding goal, with 27 days left:
> http://igg.me/at/yourfirstgmo/x/4252296
> T: @onetruecathal, @IndieBBDNA
> P: +3538763663185
> W: http://indiebiotech.com
> <0x988B9099.asc>
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