jwb wrote:Hi mchlbrmn,
I am the only person in the lab who keeps apart some ligation mix as back-up (which I regrettably didn't, this time).Everyone else uses the whole thing and they get enough colonies all the time.
One thing I feel though is that the ligation mixture has to be mixed once with the pipette before using it for transformation. On one occasion I remembered that my back-up mix(5 ul) gave me many more colonies (4-5 fold more) than my initial transformation mix (15 ul). This, I presumed, is because some DNA kept stationary for quite some time in the tube before transformation sinks down to the bottom. It sounds rubbish, really, but maybe it could happen. The competent cells I used were the same batch.
I've noticed that some ligases seem to require using alot of the ligation reaction to transform the bacteria. I always use fast-link ligase from Epicentre (CamBio in he UK), I have no connection to this company but this product works really well and it's relevant to your post so I thought I would recommend it. You can do transformations with 0.5ul of your 20ul reaction (I generally use 1ul). I usually use about 10ng of acceptor plasmid and a appropriate amount of insert for my ligation reactions, so the amount of plasmid needed to get colonies with this kit is less than 0.5 ng. That also means you always have tons of extra ligation left over in case something goes wrong.
On a side note using too much of your ligation can kill your transformation. We had a guy who couldn't get his cloning to work even though everything looked good. In the end it turned out he was using 10ul of the fast-link ligase reaction because that was what he did with a different kit in his old lab. He dropped the amount of ligation mix down to 1ul and it worked like a charm.