You can also generate the knockouts using TargeTron – Gene Knockout System by Sigma-Aldrich.....

http://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/targetron.html

thanks neelam.but have u personally tried it..I found to some elements missing in the kits to make it as a fully trust worthy option

Well I have designed primers and will do knockout using the same. I have not tried it but have discussed with those who used the same. They suggest it works well. 1st u check if any targetron site is present in ur gene of interest. U can test it. If u find b/w 4-8 sites u can go for it. And yes apart from kit, U may need few additional things, like.. a helper plasmid or Restriction enzymes (if not compatible with strain), the detail is given in the manual...