cloning problem - (May/07/2007 )
I had a problem my experiment is include 70bp in 4255bp vector
When I digested my vector by bamh1 and xba1 I got to band the large one and the small one I extraction the large one with gel extraction kit the I ligate it with the insert (cut with bamh1+xba1) the problem when I screen the vector most of the insert are small one before I hope I explain it good so pls help me
I'm sorry to say that I didn't quite understand with your last sentence. Could you please explain it properly?
Anyway, theoretically, 70bp insert should be very easy to be completely digested by restriction enzymes. However, practically, sometimes it is rather difficult to confirm that.
I would suggest that you clone your insert into a TA cloning vector (if your insert is PCR product), digest it with your restriction enzymes, and purified the digested insert from gel.
With the gel purified insert, set up your ligation with the vector. Also, make sure that your vector is completely digested.