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

Hi,

Yes, I've tried a micro-homology based approach but I must say that was a very rough try, with a gene potentially toxic, using home made chemical competent cells.
Plasmid itself was quite big, around 7kb.

I like BASIC concept, even though I'm an advocate of PCR based approach, but recently both PCRs and biobrick based cloning completely sucked. Moreover as we are looking into implementing an automated cloning system in our lab it makes total sense to shift toward an automatable system.

My feeling is that biobrick system is very expensive and, the ligation step, isn't that reliable or standardized as you use different blunt/sticky ends.
I'm pushing to use commercial cells as well.
Basically time you waste and number of try increase your end price incredibly.
Even if you use home made stuff.

So far that's what I see:
- multiple assembly in one-pot reaction
- less restriction enzymes
- avoid completely gel purification (different marker at level 0)
- highly plastic to permutation and combinations
- change adaptor when you need
- fast and cheap
- automatable

I believe it will be a pain to set everything up but it's definitely appealing.

Potentially this system has an advantage also for the diybio community.




Fede

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