TOPO >10Kb into pENTR-D-TOPO? - (Dec/05/2006 )
I am thinking to topo cloning a 10kb PCR product into pENTR-D-TOPO... does anyone have experience before? I can certainly use TOPO-XL but pENTR-D-TOPO offers more convenience for restriction sites.
I've done quite a bit of TOPO cloning and, if it works, is easy, fast and efficient. I did have a problem when trying to subclone a large insert (9.5 kb) as it would always be in the wrong orientation. TOPO is supposed to be a 50/50 for orientation but sometimes (especially with large inserts) it can take on a tertiary structure which inhibits the TOPO ligation. Only way to find out is to try it!
Are you talking about TA TOPO cloning kit if there is 50/50 orientation problem? I don't think directional TOPO will have the same problem...