opposite sequence orientation in TA cloning - (Aug/31/2007 )
I used the pcDNA3.1 V5-His TA cloning kit (invitrogen) to clone my gene. But the digestion results indicate the inserted sequence in all the 11 colonies I got are in the reversed orientation. I don't have much experience in the molecular cloning. 
Did anybody have the same bad experience? Is there anything I can do to avoid this problem? Any help will be appreciated. Thanks a lot!
-avin-
Hi Avin,
I've never used this vector before but I guess, as with any other T vector, the problem is that is non-directed cloning, so is just a question of luck that your insert ligates in the correct orientation.
I'd suggest directed cloning with restriction sites, it has a few extra steps, but generally works better.
-almost a doctor-
QUOTE (almost a doctor @ Aug 31 2007, 08:20 AM)
Hi Avin,
I've never used this vector before but I guess, as with any other T vector, the problem is that is non-directed cloning, so is just a question of luck that your insert ligates in the correct orientation.
I'd suggest directed cloning with restriction sites, it has a few extra steps, but generally works better.
I've never used this vector before but I guess, as with any other T vector, the problem is that is non-directed cloning, so is just a question of luck that your insert ligates in the correct orientation.
I'd suggest directed cloning with restriction sites, it has a few extra steps, but generally works better.
Thanks a lot, 'almost a doctor'. the invitrogen people also gave the same suggestion. I'll try the directed cloning soon and hope it will work.
-avin-