basic question - (Mar/02/2006 )

Hi

I m sorry I m going to ask a silly question but really I don't know the funda or may be I know this and I might not be getting it at this time. In classical cloning, we go for ligation of PCR and vector. After insertion we see if it is ligated or not and then we go for digestion,then again insert it into expression vector to express the protein. The question is Why don't we directly go for expression. I mean we can directly ligate the PCR product with expression vector na. Why there is so many cutting and joining?

Thanks

hi
i think two differences occurs.

Classical ligation includes a fragment and a vector (expression vector or not). You need to check it by restriction analysis or pcr-on-colonies analysis.

But sometimes it's hard to do. So A tail is added to 3' of the PCR fragment and the PCR fragment is ligated in a vector (Topo TA or TAcloning kits), and you electropore bacterias and plate them. You need to check some colonies to see if you insert is in it.
After you do a midiprep of plasmid, and by retriction digest, you can obtain a fragment of interest (actually the so called "insert") which you're sure all the ends are properly digested (what is not possible when you're digesting PCR products). Purification of the digested product ensure to get better insert. This final fragment is the so called "insert" which is ligated in expression vector.