Help with Cloning

Prema premasundaram at
Tue Sep 28 21:02:09 EST 2004

I am a graduate student and I have started doing some basic cloning. I
am doing a simple directional cloning using KpnI and Not1. What I am
trying to do is to subclone a TA cloned cDNA into a doubel T7 vector.
I cut the vector and the insert with these enzymes, run them on a gel,
cut out the desired vector and insert bands purify through a qiagen
column and then set up my ligations. When I test the identity of the
colonies that I get using restriction enzymes, all the colonies test
positive for one or both of the parent plasmids that I used to get my
vector and insert fragments. How is that possible when I cut out the
right bands from a gel and then ligate them. I usually do my digests
for 2 hrs and let the gel run long enough before I cut the fragments
out. Can you give me some suggestions. Also the kpnI and NotI sites
are in the MCS. It is really frustrating because it looks like a
simple cloning method but is not working for me!!!!

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