2 replies to this topic

[Orion]

Hi all,

Recently I had tried to clone a 1.3kb gene into pYES2 vector from invitrogen and after ligation, I transformed it into TOP 10 competent cells. However, I only managed to get 2 colonies on my LB/Amp agar plate. Later on, I did some checks and realized that my insert DNA concentration used was too low during the ligation and thus causing the undesirable results.

However, a question struck me at that moment. In the above scenario, there should have alot of cut, unligated pYES2 vector. So I am wondering whether can  these "linear" and cut vectors be transformed inside the cells? And if so, can the amp-resistence part of the vectors still function normally? If the answers to the above 2 questions were yes, then I should be getting more than 2 colonies on my plate right?

Sorry for the lengthy post, but it is a curious question that struck me and I have been searching for answers for quite some time but to no avail. If anyone can answer me, please enlighten me. Thank you in advance.  

[dpo]

Yes, the linear fragment can be transformed into the cells.

Yes, the amp-resistantce should still function normally.

BUT since your plasmid is not circular, it cannot be replicated completely. When the cell divides, only one daughter cell will have the antibiotic resistance gene ... As a result, you don't see any colonies growing if you transform a linear fragment.

[Orion]

dpo, on 10 August 2011 - 02:09 AM, said:

Yes, the linear fragment can be transformed into the cells.

Yes, the amp-resistantce should still function normally.

BUT since your plasmid is not circular, it cannot be replicated completely. When the cell divides, only one daughter cell will have the antibiotic resistance gene ... As a result, you don't see any colonies growing if you transform a linear fragment.

I see... makes perfect sense now.
Thank you very much.