No positive colony - Help (Jun/15/2006 )

I'm trying to obtain a positive clone, and I don't understand why I don't manage.
I want to put two insert of 700bp and 3300 bp in a 10kbp vector.


I do doble digest of my inserts and vector. One insert digesting with Stu1 and Hind III, the other insert with Not 1 and Hind III and the vector I digest with Not 1 and Eco RV. I do the digest with one enzyme and after 1 hour put the other one enzyme for the same time (1 h) at 37°C.
I purified my plasmid by Qiagen Kit and it´s ok
For transformation :
- 5 µl ligation product in 50 µl XL Blue calcium competent E coli cells


I obtained as many colonies for the control (plasmid only) but not as for plasmid plus my inserts.

Do you have an idea of what happen? Can you help me?

can you tell us your ligation conditions???

It can happen when the digest is not complete. U need to digest them longer than 1 hr.

so please tell me more about your digestion conditions, particularly the Not I / Hind III double-digest

am I understanding you correctly? you make up the reaction. after it proceeds one hour, you add the second enzyme to the tube and put it back in the incubator. why? you can add both enzymes at the same time and put it in the incubator for a couple hours to get more complete digestion (I really think an hour is not long enough, like Scolix said)

you are not purifying between enzymes, or fiddling with the buffer in the middle, are you?

I paid attention to their observations.......the problem I think was the time of ligation..... I leave overnight and finally today I was able to obtain a positive colony .......thx for all....

Best wishes.......
Gustavo