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…

1 comment:

  1. Ah, restriction cloning! Taking me back to the good old days of science class. Nostalgia truly hits differently when you reminisce about those moments huddled around a lab bench, pipette in hand, trying to master the art of DNA manipulation. It's amazing how far we've come since then, with newer techniques and technologies making these processes even more precise and efficient. Here's to the memories of restriction enzymes and agarose gels, and to the exciting advancements that continue to shape the world of molecular biology! 🧬🔬 #ScienceNostalgia #DNAManipulationMasters