Hi All,
Ive been constructing some plasmids with 4-12 pieces at a time pretty regularly using golden gate assembly for about 6 months now, but its getting to be a bit cumbersome due to relatively low efficiency of some of the constructs and really time consuming construct planning and trouble shooting. Someone at my work mentioned that they've had wild success with cloning difficult constructs (over 20 constructs, ~30kbp, 8-12 piece assemblies at >30-80% correct sequence) in yeast where all other cloning fails. It sounds almost too good to be true. The planning is easier, the reactions are simply TAR reactions, the only downsides i see are (1) the need to transform back into bacteria for large scale DNA preps and (2) long incubation timelines after transformation.
I was wondering whether anyone prefers doing all their cloning in yeast transfer vectors and then transforms back into bacteria? Am I naive in assuming this could save me massive amounts of time in construct planning and cloning execution?
Some interesting background info:
14 fragment assmebly of SARS-CoV2 within 3 days of receiving all the DNA fragments of interest:
https://www.nature.com/articles/s41586-020-2294-9
Low background yeast coning by transforming back to bacteria (which is what I planned for plasmid prep):
Article Ultra-Low Background DNA Cloning System
Yeast cloning kit from Thermo:
https://www.thermofisher.com/order/catalog/product/A13286#/A13286
Hi Dr. Duguay how big are your overlaps?
I havent tried it yet, but Im planning on trying it out next week. Ill keep you updated...
My plan for getting the fragments out of the yeast was to assemble all fragments in yeast with the YAC or ARS and then PCR out the relevant cassette (-) the yeast stuff, circularize, and then transform back to bacteria
- Badges
- Science topic