Hello. So for a current project I'm doing, I'm assembling several fragments (6-7) using golden gate assembly. I've combined the ligation and digestion in to one reaction, which takes ~5 hours in total. Right now, I'm trying to figure out ways to make the assembly process faster. Does anybody know a more efficient assembly program for 6-7 fragments in to a target vector? Thanks.
You could try out Gibson Assembly and/or In vivo assembly (IVA) cloning. Make sure to have proper DpnI digestion for all fragments that are very similar in size to their template (e.g. if you amplify a whole vector backbone minus MCS). Otherwise empty vector background in my experience is tough.
IVA Cloning
Article In vivo cloning of up to 16 kb plasmids in E. coli is as simple as PCR
IVA cloning amazingly really works... So far I have only tested a two fragment assembly (4.5 + 5 kb), but it was amazing enough. I am soon testing a 5 fragment IVA. *keeping-fingers-crossed*.
Gibson assembly
For Gibson assembly I prefer the commercial NEBuilder kit
(https://www.neb.com/products/e2621-nebuilder-hifi-dna-assembly-master-mix).
It has worked amazingly well for me in the past for everything from 2 to 5 fragments (did not try more) including bridging oligos etc.
My current approach is to generate fragments with 25-30 bp overlap, run them for 1 hour at 50°C in NEBuilder (20 fmol each in total volume of 5 µl DNA + 5 µl NEBuilder) followed by 10 min at 98°C for inactivation. 2 µl of this reaction I transform in high competence cells (I currently like NEB Stable best, 20 µl of bacteria is sufficient for trafo), plate all bacteria on Ampicillin plates and let the grow on the bench from Friday to Monday. This way, presumably incomplete Gibson assemblies can be further completed in the bacteria by IVA over the weekend. This way I frequently get 14 out of 16 positive clones from a 5 fragment assembly (Fragment size 0.7 to 1.8 kb).
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