I recently successfully made a plasmid using 5 parts (one of the parts was the vector backbone). I used the GeneArt Gibson Assembly® Cloning mix. Out of the 52 colonies that I screened (using colony PCR) only 1 was succefully assembled. This is 2% assembly efficiency! Is it normal?
Increase the gibson assembly reaction time to 45mints-1hr when assembling more than 3 parts.
I've noticed the efficiency of Gibson Assembly can be pretty dependent on the purity of my fragments, the length/tm of my overlaps, and the proximity of the ends to either regions with repeats/high secondary structure. Large insert fragments can also be a bit of an issue.
With higher fragment assemblies, background transformants from plasmid templates also becomes much more apparent.
Thank you Alexandra Johnson. It was interesting that I on;ly had success when I made "fresh"fragments with FI-HI PCR and used these the same day for the GA. When I design overlaps again I will keep the tm in mind. I did have suggested overlapping lenghts (for 5 pieces was 40 pb) and did take note of the secondary structure. Unfortunately when precise sequences have to be ensured at the overlapping regions one does not always have much control over these factors. Or perhaps I am I missing something obvious?
- Badges
- Science topic
- Similar topics
- Vectorization