transformation:more colonies with control! - control plate twice as many colonies as ligation (Mar/09/2007 )

Hi!

I'm an absolute beginner in cloning and have been trying to put a 5.5kb promoter fragment into the pGL3.basic vector for a couple of weeks now. So far, I haven't been successfull. I'm using a PCR product for which I used primers tailed with a MluI or XhoI site. I always got the empty vector after the transformation with exactly the original sequence between my two cutting sites (which indicated to me that one of my digestions didn't work properly). This time I used more enzyme, gel isolated vector and insert after each digestion and also dephosphorylated the vector after the second digestion (twice 0.01U CIAP per pmol DNA ends, according to Promega's manual).
The ligation went over night @ 16° and I also set up a control with the digested vector only. Now, after the transformation (5µl ligation for 50µl competent cells) I have about twice as many colonies in my control plate (vector only) as on my ligation plate (vector + insert) (150 vs. 60 colonies).
I'm confused now. Not only should the gel isolation and dephosphorylation prevent any vectors from self-ligation. But how can it be that there are more (apparently) empty vectors in the transformation of the vector alone that in the one with the insert?
Does anyone have an explanation? I would greatly appreciate any ideas!
Thanks!

couple things, maybe...

1. phosphorylation is often flaky, even when all directions are followed. everyone has their particular favorite and swears the others don't work you may wish to try another, although I suspect this is not the main problem

2. I am guessing you are adding more material from the insert than from the vector to the ligation reaction; most of us do? anyways, there can be things that are inhibitory to ligation that are present in your tube after gel purification. perhaps examine your method...use a PeOH/chl or other add'l purification on your fragments

anways, I'm going with the second as my best hunch...best explains why your control worked better all things being equal, your control and ligation rxn plates should have roughly the same amount of background, so there almost has to be something inhibitory that is coming from your insert piece

good luck

Other thing:
Do you heatinactivate your CIAP before putting your vector into the ligation (or get rid of it in some other way)? Because if you leave it active in your ligation, it can also dephosphorylate your insert in the ligation.

I've also experienced problems in cloning a promotor in a vector, and haven't overcome that. I can't find any clue about what's happening, other sequences clone easily in the same vector, so maybe it's in the sequence that gives rise to some toxicity in the bacteria. Is it a prokaryotic promotor?

many times cloning can make no sense.

As you are using MluI, I should suggest that you be careful with this enzyme. too much or too long digest can prevent proper digestion and eventually ligation.

Try to heat inacivate after Mlu1 digest and digest no longer than 1-2 hrs with appropriate amounts of DNA. i would do it for 1 hr. if you think something is not right with the batch of enzyme you are using, get Mlu1 from another company.

Mlu1 is an enzyme which has bugged me in my old lab and even when I started in my presnt lab. So I am really careful with this enzyme.

Good Luck !!!

I always figure heat inactivation sort of limbers up the strands and helps to remove any residual bits of protein stuck on the DNA. I think it's always a good idea; only takes a few minutes and it's very unlikely to hurt anything

I agree with aimikins, heatinactivation is a good way to ensure the complete removal of your enzyme on your DNA, as well as the complete inactivation of your enzyme.
While doing the clean up, you get rid of most of the enzyme, but there can still be enzyme attached to your DNA that is copurified.

Good luck with your cloning!