How do you check you ligation? via Agarosegel electrophoresis? Could it be that your insert is toxic or something like that? Otherwise you may try it again with other chemocompetent E. coli from your lab. I used to use DH5alpha and it worked very well together with Gibson assembled constructs.

Did you use the positive control given in the kit? If that works maybe something is wrong with your assembled product or the competen cells from the kit and you will be able to check your method is not going wrong.

But just guessing what was spontaneous in my mind. Hope you will be able to fix he problem.

I'll keep my fingers crossed for you.

Sara

thanks sara, already i do transformation vector only with DH5alpha and transformation done but when i do transformation with comptenet cells provided  in kit no grow. however  positive control provided in kit is work but mine reaction not done in transformation.

about how i check the ligation reaction on agarose gel iam just do it  and after that run 5  microlitter comparing with vector only without fragments  very weak bands appaear at expected site.

i think may be you are right some thing not good in comptenet cell provided in kitor what you advice me do.

respect

doaa

you are doing right checking your ligationin agarose gel as you do .

If i understand it right you are now using DH5alpha to do transformation of your assemled product and there are stil no colonies.Right? But transforming your empty vector/positive control from the kit you get colonies using DH5alpha.

In the case you can be sure your Gibson assembly worked (as you checked it on Agarose Gel) there must something different be wrong. But now you can exclude the competent cells. 

how big is your plasmid? is the insert e.coli specific or is it from an other organism? if you like you can tell me what the insert is i have some persons to ask for help. Because there is still the idea, that the insert is kind of bad/toxic for your e.coli and they are dying because of that, and not because the transformation doesnt wort. you know what i mean?

I never had problmes with gibson assembly, so i am an kind of surprised. If you can not solve your problem try to contact the NEB Support. I read in the internet, that they should be very kind.

Greetz Sara

helllo sara

positive control provided in kit not give me colonies, also transformation my vector only in comptenet cells provided in kit does not work but with DH5 alpha work. 

so, i think the problem in comptenet cells. but my plasmid with 2 fragments and not exceed 10000 bp.

also insert not toxic i check that.

many thanks

yours

doaa

so keep me up to date if you can totally fix your problem.

Good luck and take care!

Sara

ok, thanks sara

doaa

no ya sara  abdou  this not the problem assembly very special technique i solve the problem already , merci

Sara Werner many thanks i do your advice in last massage , design primers with more overlap bases, it is working now