For the Gibson cloning into pH-ePPE vector (19kb), I use NEB Hifi builder mix with 400ng of vector backbone (18kb) and 10ng of 250bp insert and NEB chemically competent 10beta cells for transformation. I know my Gibson assembly is working as I have confirmed by PCR. I have used 1ul to 10 ul of Gibson product as well as 1ul of 1:3 diluted product, but I am not getting a single colony post transformation.
- The competent cells are functional, verified by transforming the vector pH-ePPE.
- The vector doesn't have any toxic genes like ccdB and I also confirmed that the gibson mix is not toxic to cells by using positive control.
- I also used NEB 5 alpha cells, but no no colonies with that also
Can anybody suggest how to troubleshoot this problem.
Shiksha Kotikalapudi
Thanks for your suggestion, well appriciated.Regarding the NEB 5 alpha cells, I have checked it as well as Gibson mix with the positive control, everything is fine.
About the insert:vector molar ratio, NEB suggests not to go beyond 2:1, so we didn't tried it, can you please tell me whether you have tried those ratio (5:1 and 10:1) and if so, how was its effeciency.
If you are confident transformation and selection are not the problem then Gibson must have not worked.
When you check your Gibson with PCR, are the primers binding one to the insert and the other one to the vector? Otherwise, you will get many false positives.
If you are using the right primers, I would also run a negative control PCR just with your vector and another one just with the insert to check they are not binding non-specifically (although it seems unlikely that if they bind non-specifically they would produce the expected band on a gel, I've seen it before!)
If you amplified it using appropriate primers my guess is that somehow it has ligated on one end but not the other, thus being a linear product that can amplify but is not able to transform cells. Maybe you could use another pair of primers that bind to the other insert-vector end you are not checking and see if that one amplifies.
Trying new ratios seems sensible,
Good luck!
I also have the same problem. I tried using 1:3 , 1:5, 1:10 ratio but still. In 1:5, I got colony, but when I checked, it doesn't have my insert gene. I'm wondering why too
Hi Sanjay T D I'm currently trying to clone a 700bp insert into a 18kb vector and I'm having the same issue as you : no colonies. I'm pretty sure the problem comes from the transformation part and not the gibson assembly. I've read a lot about it and apparently 18kb is really big for bacteria if you're doing a heatshock (like I do). people recommand using electroporation instead (if you can).
If you manage to clone your insert please let me know because I'm really struggling. So far I've tried different ratios 1:1, 1:3, 1:10: 1:20 with vector quantities from 100ng up to 300ng.
Have a nice day.
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