cloning problem of big inserts - (Jul/16/2007 )

hey,

i try to clone one big insert (5,8kb) into a quite big plasmid (12 kb) and so far without any success. The insert is PCR amplified, cloned into pGEM-t easy and at the end XhoI digested. The plasmid is linearised with XhoI as well and in theory it should work. I deP the vector and at the end I get a huge number of colonies (more than on the control plate) but none of them contains my insert. I use the Roche rapid ligation kit and our competent bacteria stock is OK and the previous 2 cloning steps were easy. but after overnight incubation the bacteria just contain some kind of junk DNA and not even the self-ligated destination plasmid. I have no idea why and it could happen.
Somebody has any idea?
Thanks!!

I have some experience with cloning big insters into big vectors (mostly about 5 kb into 10 kb plasmid).

What enzyme did you use to deP you vector? Phosphatases aren't nice to DNA, though Antarctic from NEB was good to me.
You have no possibility to use 2 different restriction enzymes?

My suggestion would be to gel purify insert and vector. Use 0,7% agarose and let them migrate LONG (3-4 hours) at lower voltage to get nice separation. Try to minimize exposure to UV.

Grow bacteria at lower temperature (helped in my case, I have repeat sequences in the plasmid).

aside from growing your cells at a lower temperature, I would strongly recommend that your buy yourself some company made competent cells. My choice would be Genehogs® (invitrogen) electrocompetent cells.

My feeling for this situation is that your ligation efficiency is not good enough, further complicated by the large size of the plasmid. You need good competent cells to get this plasmid, and the home made cells may not be up to the task.

The Genehogs cells grow slowly though, and you may need to grow them for 20hrs or so.

Thanks for the tips. I use CIP from NEB and before it worked every time. We have the other enzyme as well so I could try. I use TOP10 bacteria from Invitrogen which works always so it can't be a problem. Maybe I try the lower T. This would mean 32 or lower?
XhoI is my only possibility cause it's a difficult vector and I don't want to introduce another restriction site into it.
What starting amount would you use?
thanks again.

I also used TOP10, chemically competent. I don't remember the exact amounts I used, but I used 5 µl of a 20 µl ligation reaction performed according to Invitrogen's T4 DNA ligase manual.

I grow @ 30°C, but 32 might also work.

Do you gel purify your insert?