Re: [DIYbio] Re: The ODIN Needs Your Help - T4 DNA Ligase Testing - Free Ligase

This reminds me of a goldengate PaperClip assembly. Very interesting however, once I get an opportunity I will read the entire paper! 

You tried the DH5a method or the SLiCE method?

-Koeng

On Monday, October 19, 2015 at 3:42:46 AM UTC-7, BraveScience wrote:

Same here. Ligation dependente cloning using biobricks sucks, given the volumes and the amount of steps you have to necessarily go through, doesn't turn out to be that efficient. 

I got interested as well in Gibson. Indeed price\quality of commercial kit vs homebrewed one is a tough one.

Anyway I stumbled across the BASIC system. Yes, you need a restriction enzyme (just one, BsaI) and work through ligation. But you can shuffle your parts around as you wish.

http://pubs.acs.org/doi/abs/10.1021/sb500356d

It's a one-pot reaction.

Fede

Il giorno sabato 17 ottobre 2015 22:14:42 UTC+2, Koeng ha scritto:

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0137466 

May be interesting

On Saturday, October 17, 2015 at 1:09:32 PM UTC-7, Koeng wrote:

http://www.sciencedirect.com/science/article/pii/S2405580815000850

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4453199/pdf/12896_2015_Article_162.pdf

 It appears that JM109 can do SLiCE just fine. Not sure about comparisons of efficiency thou

-Koeng

On Friday, October 16, 2015 at 9:14:10 AM UTC-7, Bryan Jones wrote:

Koeng, SLiCE sounds interesting. Do you know where to get your hands on the PPY E.coli strain that they use? Is it sold anywhere (a quick google search didn't turn up anything)? I guess I could try to contact the authors or those papers. I'd like to give it a try in the academic lab I'm working in. If it works as well as they claim it might be a good alternative to both traditional restriction enzyme/ligase cloning and Gibson Assembly, especially for the DIYbiologist on a tight budget.

On Friday, October 16, 2015 at 8:27:08 AM UTC-5, Koeng wrote:

True true, EcoRI and HindIII are favorites of mine because of how much they supply. However, they are 6bp cutters... So I would actually argue that NotI and SbfI would be better for a new cloning system! I guess it depends on how much it would cost to remove the sites vs the actual enzyme cost.

I've noticed that at least at an academic lab people always use the same volume of enzyme... Like 1µl is 20 units of EcoRI but just 5 units of BtgZI... anyway it's a pretty wasteful approach, so if anyone is reading this is using restriction enzymes, go by units.

Then again, I haven't even cloned anything with classic restriction enzymes in a year or so because gibson is so good. However, gibson can cost an arm and a leg if you buy it directly from NEB (price for quality, their stuff gives me ~5-10xs better efficiency than our normal stuff). I know that SLiCE ( http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3333860/ http://www.ncbi.nlm.nih.gov/pubmed/24395368 ) would probably be very cheap to make in a DIY lab if created in mass, which might be useful for you. I'm curious what the efficiency is, but if it's good, I'd be willing to switch my home experiments over if it meant I wouldn't have to buy mixs anymore.

-Koeng

(btw: Thanks! I knew I forgot something)

On Thursday, October 15, 2015 at 10:27:12 PM UTC-7, Josiah Zayner wrote:

Yeah it is just a resale. Most people don't have ATP I imagine so I want to make sure it works as is, ya' know?

I will look at BsaI BsmbI but they are less used enzymes so they are freaking expensive.

You can do like 10 times the cloning with NcoI/EcoRI/HindIII on price maybe even more.

Same thing with Biobricks enzymes. I am selling those for iGEM folks but they are expensive. Someone should go back and start a cloning system that uses EcoRI and HindIII, the two least expensive enzymes.

Also, Koeng, you forgot to use the coupon code so I refunded you shipping!

On Thu, Oct 15, 2015 at 10:14 PM, Koeng <koen...@gmail.com> wrote:

How about the raw ligase? I buy ATP directly from NEB to do goldengates with, and some ligase from a non-commercial source might be interesting. 

Also, how are you getting the ligase? Expression of it in house? Or resale? Either or, I'm sure it'll be a great offer, but I am curious if you had the vector in house for some in vivo ligations I'd like to test out. Anyway, useful site! It's awesome that you've gotten more enzymes up, but I think BsaI/BsmBI are also a real important couple. Once you get materials to do GoldenGate, it's a pretty wonderful system, just wish I could get around to making some basic parts for DIY vectors...

-Koeng

On Monday, October 12, 2015 at 1:37:02 PM UTC-7, Josiah Zayner wrote:

My company, The ODIN(http://www.the-odin.com) is starting to ship restriction enzymes and ligase! However, enzymes are pretty heat stable. T4 DNA ligase is another matter, it requires ATP, ATP hydrolyzes. I need a few people to test out some T4 DNA ligase and reaction buffer that has been shipped. Shipping of Taq and other Master Mixes has worked and they contain dNTPs so I assume this should work out just fine but I need testers to make sure.

If you goto the website(http://www.the-odin.com/t4-dna-ligase-5ul/) and use coupon code FREETHELIGASE I will ship you some ligase for free as long as you promise to provide feedback on how it works after shipping.

Thanks,
     Josiah Zayner
     The ODIN
     ca...@the-odin.com

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