Yes, cloning itself is a not-so efficient process. It is very easy to have an E. coli take up a complete, circular vector. But the process of creating a plasmid, i.e. to ligate an insert correctly into a vector, is much less efficient. You are not making a mistake, you are describing a very normal difference in efficiency. There are many tricks in getting the cloning reaction to be more efficient, but this is very dependent on your strategy. You can work with sticky-ends (e.g. EcoRI digested fragments) in certain cases, or you can work with TA vectors (e.g. pCR4 TOPO TA from Invitrogen) to clone PCR products. Gel purification of DNA fragments before cloning usually improves efficiency as well.

I hope this is helpful!

Thanks alot for your kind reply, i will try to follow your suggestion...........

I agree with Elisabeth and one can probably write a dozen more suggestions. I will add a few. Transformation efficiency is dependent on many variables, even the transformed sequence. But obviously in most cases you are stuck with the sequence you have. When designing your construct try not to have large repeats or large stretches of purines or pyrimidines (Like typically AT rich plant stop signals would be difficult to keep stable). Try to develop a cloning strategy in which a unique restriction site disappears, it allows counter selection by cutting your ligation mix. If really bad sometimes cleaning up your ligation mix. Or switch to electroporation instead of traditional competent cell transformation.

thanks for yours kind answers...... this time i have purified the DNA fragment via gel and hoping to get sum good results.............

in cloning you have to think about DNA concentration in terms of molar (mostly picomolar), not in µg/ml. You should play with the concentrations in your ligation mix in function of the self ligation capability of the vector plasmid.. Keep in mind that only cells receiving vector will show up. So for blunt end ligation or compatible sticky ends on the vector give at least 10 x more insert (on molarity) so that you push the reaction towards insertion of your fragment. This is much less important when your vector can't ligate on itself.

Ligation is step before transformation. You will get more colonies if your ligation is efficient. Sometimes, the RE digested plasmid which has been self-ligated also gives some colonies, which are negative.

Also, the amount of ligation mixture used also determines transformation efficiency. Usually, transformation efficiency is higher with 2-5 ul ligation mixture, which is sufficient enough to get good number of colonies.