ligation: only false positives. Advice needed - (Oct/02/2006 )

Hi
I am working on 4 enzymes and I have to put them into pET15b.
Their sizes are 698bp, 720bp, 930bp and 1250bp. The vector is about 5100bp
I put them all into TOPO TA2.1 vector and the sequences of my inserts have been checked.
But then the problems started.
I first had a lot of false positives. I use pET15b with XhoI/BamHI (sites are close) for 3 enzymes and decided to use pET15b with an insert in the NdeI/BamHI for 1 enzyme.

The false positives were due to uncut or partially cut vector. So I use CIP (after gelpurification with QiaQuick) for 1h at 37°C to remove the terminal phosphatases of my vector.

But now other problems have occured. Although my ligation control of vector and of insert (to make sure it is not contaminated) is good, I still get false positives.
I already tried different insert:vector ratios, different ligation times but it still gives the same result.
These false positives can sometimes be identified as vector ligation, but most of the time I just see unknown bands ranging between 800bp and 3000bp. In most cases, only 1 band is visible.
Since they have different sizes, it can not be due to a contamination of one product.
I just don't have a clue where these bands come from.

The only thing which might cause this is the presence of other stuff in my insert or vector digest after purification (although invisable on gel before ligation)
I'm trying a digest today, and I will use CIP only with the half of my volume to check its influence.

A common source of unknown inserts is carryover of genomic DNA into your plasmid prep. When cut with your restriction enzyme, this genomic DNA becomes a source of active inserts into your cloning site. You can minimize this problem by careful lysis and gentle treatment of the genomic DNA during miniprep processing, and by not overloading the prep with to many cells.

It sounds to me as if your double cutting of the target vector is problematic. You should have low background with a double digestion. The high background suggests that you are failing to cut with one or the other enzyme, perhaps because they are too close to one another. CIP will prevent religation, but will also trash the ends of your DNA when overdone. If possible, I would look for another pair of enzymes on your vector.

First I want to mention my digest of vector is OK now, I use vector with an insert for one digest and I do a sequential digest for the other combination. I also once checked if my vector was cut by adding 1 enzyme and putting a sample on a gel to make sure all of the vector was linearized.
Yesterday I've checked all my plasmid DNA and my digests ware fine. I will have to prepare new plasmid DNA to avoid genomic DNA. I know steps 2 and 3 of the midiprep were done in the right way but I use more culture than I should. Let's hope this is the cause!

I'll stay in touch.