Transformation problem with newly ligated plasmids - (Sep/10/2007 )

I am trying to ligate my pCS2 plasmid (~3.8kb after double digestion) with two other sticky DNA fragments (one is
~500bp and the other is about ~1300bp in size), which is a tri-molecular
reaction. The two different DNA fragements are actually PCR clone products with sticky ends and they can ligate with each other in the middle.

I use the NEB quick ligation kit and follow the instructions for ligation:
ligation at room temperature (25 degrees) for ~15 minutes. With the 'newly
ligated' (hopefully) plasmid, I transform it into super competent cells
with a 'negative' control where only the open vector is used. However, the
problem is I also got a few colonies from this negative control. My thinking
is:

1. The open vector is circularized in E. coli?
2. or the open vector is mixed with parent plasmid that is not digested
completely.

As of the above 'open vector', I cut my parent plasmid with two restriction
enzymes (Cla I & Xho I, with NEB buffer 4), which is followed by gel
extraction and purification.

When I run the 'open vector' along with the uncut parent plasmid, they run very closely, which is kind of hard to distinguish. But I do see the ~500bp fragment that is cut out of parent plasmid. I guess the supercoiled parent plasmid runs faster than linear DNA of the same size. Considering my linearized plasmid is ~500bp smaller than the parent plasmid, they probably run the same. My question here is: how to separate the linearized plasmid and the supercoiled parent plasmid better? lower voltage and/or longer running time?


ps: My LB media + Amp is good for selection.

Do you guys have any suggestions/comments?

I appreciate your help.

Hi,

you say you get "a few" colonies in the negative control. How many do you actually get, and how many colonies do you get from the actual ligation? ... if it is really just a few colonies, it may not be a problem.