Hello! I have ordered a plasmid in 5 fragments with comatible ends when cut with BsaI and my aim is to transform E.coli DH5a.
Golden Gate Reaction performed with Biobrick Assembly Kit of NEB has not succeeded yet and I am wondering if other methods should follow the linear ligation in order for the ends to stick. I read a protcol where T4 DNA Ligase is used with an incubation at 20oC for 10 minutes.
Do you think after the ligation and DNA cleanup, this could be helpful? Have you worked something similar? Any help would be greatly appreciated!
Dear Marianna,
I have performed several Golden Gate Assemblies, following the manual protocol described in the following paper. So far, it worked for me perfectly. You may have a look.
Paper: PMID: 31634902: Search-and-replace genome editing without double-strand breaks or donor DNA
Method: Supplementary Note 3: Page 35-37
https://static-content.springer.com/esm/art%3A10.1038%2Fs41586-019-1711-4/MediaObjects/41586_2019_1711_MOESM1_ESM.pdf
Good luck
Chamara
I wil study it thoroughly and try to apply these tips to my asembly too! Tank you so much Lahiru Chamara Weerasinghe Arachchige for the precious help! Cheers!
Dear Marianna,
I´ve designed and performed hundreds of GG reactions, also assembling some plasmids consisting of 6+ parts from scratch- but never used or heard of this pre-incubation step at 20 °C.
In a tough case as yours (many fragments) I would recommend to run it overnight and let a cycle a bit longer:
37 °C for 5 min (99x cycles) for BsaI digestion
16 °C for 10 min (99x cycles) for T4 ligation
Good luck :)
Best regards,
Pascal
Thank you Pascal for the advice! The reaction will last at least 25 hours but I ll have for sure a better chance this way!
- Badges
- Science topic