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Techanical Question about cloning of gene - (Apr/11/2008 )

I want to clone the gene with the restriction sites on the end of the fragment while the same sites are also present in the gene as well, but with out changing the sites in the DNA, does its possible to clone the gene through some method,
synthesis, terminal transferanse or, some over hanging method.


You could PCR your fragment and TA clone it. But how does having the restriction sites at the end help you if you have some left in the middle?


You can do a partial digest i.e. digest with less enzyme for less time. Then purify the larger (or correct sized) fragment.
If there are multiple restriction sites in the gene (>2) then this won't work.

Alternatively, as Phage said, amplify your gene but use the primers to create a unique site on the end of the fragment so you can insert into your vector. The Fermentas website used to have a great description of how to do this, using rare enzymes to help create standard restriction sites for standard vectors.