Good vector for genomic library - (Dec/17/2008 )
I need a good vector for blunt end cloning. Basically, I shear the DNA (physically or with enzymes) and repair with T4 and sometimes other repair enzymes. Sometimes I size select the fragments, eluding from a gel between .5-5kB fragments. I have used TOPO TA, Zero Blunt, and pUC19 with very little success. These vectors are mainly for PCR products. To ligate, you need to have overhangs produced from Taq after PCR. I managed to run my fragments through one cycle extension, and then ligate into TOPO. Success rate about a fourth of my 100+ clones have vectors with inserts. So my question is: Which vector is best for this type of library? Zero Blunt is troublesome, I can not get the control to ligate/transform properly. I have heard of pGEM-easy and TOPO XL. Invitrogen said they will replace my Zero Blunt kit for free or give me another vector. Any suggestions?
You may want to look at the kits available from Epicentre. Read their manuals, at least, on cloning sheared DNA. They may have some vectors which would be better than the ones you are using.
Thanks, I'll look on the web site. We have a 350 dollar credit to Invitrogen, do they make a good libarary plasmid or kit?