TA cloning, sequencing results are vector self-ligation while bacterial PCR iden - (Mar/26/2008 )
Hi everybody, I have done TA cloning these days. I found some sequencing results are vector self-ligation while bacterial PCR identification was correct. The primers used for Identification blong to insert DNA itself. I am so anxious to got some advise from you.
-IPS-
Even with TA cloning, it's normal to get a low background of vector self-ligation. If your TA vector is compatible with blue-white screening, then the colonies that contained the self-ligated vector should have been blue, under appropriate selection.
If you have other colonies that contain vector+insert, then pick one of these.
As for the sequencing, generally it's better to use primers that anneal to the plasmid rather than to the insert; if that's possible of course.
-f2dU-
QUOTE (f2dU @ Mar 27 2008, 02:31 AM)
Even with TA cloning, it's normal to get a low background of vector self-ligation. If your TA vector is compatible with blue-white screening, then the colonies that contained the self-ligated vector should have been blue, under appropriate selection.
If you have other colonies that contain vector+insert, then pick one of these.
As for the sequencing, generally it's better to use primers that anneal to the plasmid rather than to the insert; if that's possible of course.
If you have other colonies that contain vector+insert, then pick one of these.
As for the sequencing, generally it's better to use primers that anneal to the plasmid rather than to the insert; if that's possible of course.
I used M13 for sequencing, and primer for bacterial PCR is insert DNA itself.
-IPS-