| | Re: 5kb gene cloning
A very common problem here has been loss of purified PCR product, and digested vector (if using that method), on those darned kit mini-clean-up columns, if you follow the protocol that comes with the kits. If one is no longer losing most of one's DNA before even starting the ligation/cloning, this really helps the cloning, and you don't need such super-efficient competent cells. The general tip I woule give it to introduce the DNA to be purified much more slowly onto the column, in the binding buffer. Spin much more slowly at the initial stage, wash twice rather than once and leave the column to air dry for a few minutes at the end (and until you can't smell alcohol any more) before eluting the DNA, and to let the elution buffer soak into the column for a good couple of minutes before eluting. This gives great yields and is still pure enough to clone, ABI sequence, transform etc.