Restriction cloning nostalgia

Tuesday, 17 May 2016

Restriction cloning nostalgia

Today I was reminded of an invaluable table that was hung on the wall of every lab: the NEB buffer compatibility chart.

My favorite buffer is buffer 4.
Professors all to often reminisce of the days before genome sequencing, gene synthesis and proteomics. I thought it was a defence mechanism to against young researchers who take the magic for granted. Yet today I found myself thinking nostalgically about the NEB chart.
Restriction enzyme cloning is much slower than Gibson assembly, but gives a larger transformation efficiency. For libraries they have the same efficiency as USER cloning, but have the added fun/hell of the design —it is rather subjective. There is a bit of an arcane art to it. NcoI and NdeI placed right at the ATG, kpnI snuck inside a glycine and some MCS have a sequence that is packed with restriction sites right in the middle of a lacZ.  The best reminder of this world are the many big plasmids have weird and wonderful bits of debris, like a bit of a β-lactamase or transposon or a 2 kb origin —cool fact: the cassette of the KEIO collection is half junk and is filled with bits of transposon ORFs. It is definitely a dying world. Having said that, crusty profs scoff at the NEB compatibility chart: they had to prep their own restriction enzymes…

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