Rearrangement in plasmid? - (Jul/25/2008 )

Hi all,

I'm trying to clone an small insert (1.3kb) into an expression vector (5.1kb) for expression in drosophila cells. I've done the ligation, transformed into DH5a and grown overnights as per normal, and colony numbers on controls and actual ligation plate looked fine. However, every colony we picked had the exact same digest profile, 2 bands that looked totally wrong. It seemed weird that almost every colony had this, so instead of screening by miniprep and digest, we did pcr screening of the remaining colonies, and found a number of positives (~20%). Brilliant, so we grew these up (they were streaked onto another plate before PCR obviously), miniprep, and again the digests came back with this stupid two bands that were not right.

I've been told that rearrangements can occur, although DH5a should be quite good at avoiding this, and we do alot of cloning and haven't really had this problem before. I'm trying to transform into the only other strain we have XL-1 blue, and growing at a lower temp with larger volume, but any other ideas that might help? The insert we're putting in is GFP and a 3'UTR of a drosophila transcript, both of which I have cloned before sperately in DH5a with no problem, and the vector I'm cloning into has been used in DH5a loads of times with few problems. Any one got advice on dealing with this sort of problem, or have an alternative reason other than rearrangement?

Thanks in advance

Robin

Are your assumptions correct? Might there be extra restriction sites that have not been anticipated?

Could the insert be the wrong way around?

Have you tried using different restriction enzymes?

Is this insert derived from PCR amplification? Might there be an error during amplification, ie the insert has been truncated.

Hi,

Thanks for the reply. I've done the digest a number of times with different enzymes with positive controls, and the digest exactly the same. And every sample i digest is identical on the gel. The insert and vector are both just purified fragments I have cut out of existing clones I have made, which have been sequenced and they're fine. The cloning is directional and the digested bands are the right size before ligation. I've also used another cell line and that makes no difference. I'm thinking maybe its contamination with other DNA during the transformation? Or a rearrangement that prevents its from being cut?

I'm baffled.