clone - how to clone an unknown gene (Sep/09/2003 )
I am going to do clone an enzyme gene on an unknown bacterial genome,I have refered some literatures and found two methods,one is transposon-tagged,the next is useing a certain restriction enzyme to cut genome,so we can get some fragments and then link these fragments to plasmid and transfer them to E.COLI.So I want to get some information about these two methods,and if you have some better methods,can you tell me?thank you.
Try degenerate PCR, align all known protein sequences for that gene from related bacteria then use CODEHOP to design primers. Carry out PCR with these primers, clone (into pGEM-T) and confim the PCR product identity by sequencing.
There are some releveant links here: http://www.bioinformatics.vg/biolinks/bioi...520Design.shtml