I have some problem with Gibson assembly reaction. I work on viruses, and virus genome do some secondary structures and hairpin sometimes. As we have a only reaction at 50°C for Gibson assembly, I wonder if someone use a product or different condition to avoid these rearrangments! Thanks!
Dear Amandine,
I think there are several ways to tackle your problem.
1. Move the overlapping region away from the hairpin region if you have the flexibility to do that. If the hairpin is not close to 5' or 3' end, it has lesser chance to chew by exonuclease and become single stranded. By then it can prevent the hairpin formation during single strand phase.
2. Aside from Gibson assembly, there are other methods to join two pieces of DNA fragments together, for example, overlap extension polymerase chain reaction. Although overlap extension polymerase chain reaction is not that convenient compared to Gibson assembly, you can adjust the annealing temperature with this method. I think if you increase the annealing tem you can denature the hairpin structure.
Hope this helps!
Dear Donald,
1. Usually I check if there is an hairpin in the region but...here It's the problem...I can not move away from these region, I have no choice in that case.!
2. I try a fusion with PCR but it didn't work, maybe because my fragments are long (7-10 kb).. I also used InFusion cloning but it didn't work too (same problem as Gibson I mean).
Which Taq do you use for overlap extension PCR?
Thanks for your help
Hi,
i made a drawing and prose a solution in there. i would go for another entry site which is easier. You can can prolong your existing DNA fragment by another fragment so that you can use the other entry site and contain all sequences inbetween. Use fusion PCR for that as Donald proposed. or just order it as gBlock from IDT.
You could order several DNA fragment from IDT (integrated DNA technologies) and just do the Gibson with several (up to 4 fragments in total is fine) DNA fragments.
Feel free to modify my drawing if you want to present your problem better or if I understood it not correct.
You could also try to add some DMSO (up to 3%) and see if it improves your issue (for PCR it is fine)
Good luck.
Simon
Hi Amandine,
Normally, I used NEB Q5 polymerase for such long fragment to ensure fidelity. I tried to amplify 6-7 kb gene using this polymerase before, it worked for me. However, due to the length, you would expect low pcr yield.
Thanks Simon for your help, I would study your proposition.
Thanks too Donald for your help. I 've already use Q5 too.
Dear Dr.Amandine
You can check some references 1
https://academic.oup.com/nar/article/43/7/e49/2414304
2
https://www.neb.com/-/media/catalog/datacards-or-manuals/manuale2611.pdf
4
https://lib.dr.iastate.edu/cgi/viewcontent.cgi?article=6269&context=etd
https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=11&cad=rja&uact=8&ved=0ahUKEwie2MrI84_bAhUJwxQKHUMgA8o4ChAWCCYwAA&url=https%3A%2F%2Fbrage.bibsys.no%2Fxmlui%2Fbitstream%2Fhandle%2F11250%2F2351637%2F10598_FULLTEXT.pdf%3Fsequence%3D1&usg=AOvVaw2Kemp4B9Z3ec3zb0ptgMh5
11
Article Polishing the craft of genetic diversity creation in directe...
12
https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=15&cad=rja&uact=8&ved=0ahUKEwie2MrI84_bAhUJwxQKHUMgA8o4ChAWCEAwBA&url=http%3A%2F%2Fscienseed.com%2Fclients%2Ftomellis%2Fwp-content%2Fuploads%2F2015%2F08%2FNucl.-Acids-Res.-2013-Casini-nar-gkt915.pdf&usg=AOvVaw2CP2M3QG-g1d1vjSCkfwHA
13
https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=15&cad=rja&uact=8&ved=0ahUKEwie2MrI84_bAhUJwxQKHUMgA8o4ChAWCEAwBA&url=http%3A%2F%2Fscienseed.com%2Fclients%2Ftomellis%2Fwp-content%2Fuploads%2F2015%2F08%2FNucl.-Acids-Res.-2013-Casini-nar-gkt915.pdf&usg=AOvVaw2CP2M3QG-g1d1vjSCkfwHA
14
https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=15&cad=rja&uact=8&ved=0ahUKEwie2MrI84_bAhUJwxQKHUMgA8o4ChAWCEAwBA&url=http%3A%2F%2Fscienseed.com%2Fclients%2Ftomellis%2Fwp-content%2Fuploads%2F2015%2F08%2FNucl.-Acids-Res.-2013-Casini-nar-gkt915.pdf&usg=AOvVaw2CP2M3QG-g1d1vjSCkfwHA
15
https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=15&cad=rja&uact=8&ved=0ahUKEwie2MrI84_bAhUJwxQKHUMgA8o4ChAWCEAwBA&url=http%3A%2F%2Fscienseed.com%2Fclients%2Ftomellis%2Fwp-content%2Fuploads%2F2015%2F08%2FNucl.-Acids-Res.-2013-Casini-nar-gkt915.pdf&usg=AOvVaw2CP2M3QG-g1d1vjSCkfwHA
6
https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=15&cad=rja&uact=8&ved=0ahUKEwie2MrI84_bAhUJwxQKHUMgA8o4ChAWCEAwBA&url=http%3A%2F%2Fscienseed.com%2Fclients%2Ftomellis%2Fwp-content%2Fuploads%2F2015%2F08%2FNucl.-Acids-Res.-2013-Casini-nar-gkt915.pdf&usg=AOvVaw2CP2M3QG-g1d1vjSCkfwHA
17
https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=15&cad=rja&uact=8&ved=0ahUKEwie2MrI84_bAhUJwxQKHUMgA8o4ChAWCEAwBA&url=http%3A%2F%2Fscienseed.com%2Fclients%2Ftomellis%2Fwp-content%2Fuploads%2F2015%2F08%2FNucl.-Acids-Res.-2013-Casini-nar-gkt915.pdf&usg=AOvVaw2CP2M3QG-g1d1vjSCkfwHA
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