hi,

i have been trying to clone a fragment into pGEX6P1 vector. every time i amplify the insert (1.5kb), digested it with BamH1 and XhoI (sites flanking the insert), digest the vector too (since it has a 200bp fragment cloned in it, upon digestion i see the release), ligate the digested vector and insert ( in ratios 3:1, 5:1 and even 8:1) and transform it to DH5 alpha. but i fail to get insert release of proper size whenever i confirm positive clones. what appears strange to me is i see proper amplification in PCR, colony PCR and even the pcr using plasmid from one of the colonies as template but i never see proper insert release. what is possiblly going wrong? i use cutSmart buffer for all digestions.  increasing or decreasing the time for digestion does not rectify the problem too..