A Detailed Look at Gibson Assembly

[@HarveyDub14]

I've watched so many Gibson assembly vids and this is the first one that I understood. Thank you!

[@SnapGene]

You're welcome, glad it was helpful.

[@theanonymous5065]

Thank you so much! I can understand what I try to read on the internet for like 3 hours in less than 3 minutes.

[@SnapGene]

Glad we could help

[@omarsanchezjimenez6371]

How do you control how many bases are chewed by T5 exonuclease? I don´t understand why the exonuclease doesn't digest the whole strand

[@SonVu-ju5ko]

Probably T5 exonuclease has been engineered/modified to selectively function (let's say to choose a designated number of bases at 5' end), so are the DNA polymerase and ligase in this system.

[@Pelikan1490]

I think you are regulating this by the incubation time. ~15min should be enough to chew 15-20bp overhangs. If you let the reaction run for several hours, then you are right and probably will digest the whole strand

[@SonVu-ju5ko]

How do you justify primer design for short (0.1-0.5 kb) and long inserts (8-10 kb)? And what about medium inserts of 0.5-8 kb?

[@rukayatjimoh4944]

Thank you but I still don't understand how to create the overhangs on multiple fragments using snapgene.

[@SnapGene]

Hi, we have a video called Gibson Assembly with Multiple Inserts in SnapGene, you can find it here kzbin.info/www/bejne/n56nmH-mpqafetk I hope that helps.