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Ok ... so we've now tried this and sequenced verified the results. It works perfectly (and at the same rate as with the NEB HiFi Assembly kit in our hands). If you're cloning with Gibson, stop adding the snake oil and try it for yourselves :)https://twitter.com/OMarshall_lab/status/1192786148705198080 …
Technical details ... Vector backbone: 10kb vector cut at two adjacent RE sites, treated with Antarctic phosphatase, then PCR cleanup column purified. Insert: 531bp gblock, 30bp homology at each end. DH5α were transformed directly with vector and insert mix (~1:1 equimolar)
I cannot believe how much money we've wasted on kits that effectively did nothing (or at least, did nothing that the bugs couldn't do all by themselves for free). It's just crazy that this has been known about for almost 30 years, yet ignored :(
You can also do a stupid simple protocol of just lysing the cells and using that as a drop-in replacement (I like it more cause then I can use any strain). I wrote about it here a couple years ago https://groups.google.com/forum/m/?utm_medium=email&utm_source=footer#!searchin/diybio/Slice/diybio/bK89VsoF3gI …
This is crazy. I’m looking back at all the “failed” Gibsons I threw away during my PhD because the no enzyme control had high background 
It works insanely well for site-directed mutagenesis! Simply PCR your vector, treat 1h with DpnI, then transform into dh5alpha!
We have been doing Gibson-free seamless cloning in @slschmid_lab for years! Glad to see it’s catching on!
I have used PCR+dpnI strategy for some time and it went quite well. This protocol can be used to induce any sequence change < 300bp (designed in the primer region). Sounds like one-fragment Gibson. However it doesn't work for >1 fragments in DH10b.
Does anyone have experience with plasmids that contain repetitive elements, eg gateway sites, LTR sites (lentivirus), loxP etc?
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