Help with TOPO TA cloning – too long or too many insert - (Dec/07/2007 )
Hi, I really need some help. I’m doing TOPO TA cloning, with the pCR 4 vector on Bacteria, Archaea and Fungi 16S/18S rDNA and got some suspicious results. Before cloning, the inserts are amplified by PCR (Taq polymerase), gel purified (so the insert is suppose to have the right length) and added an A-tail.
The Bacteria results are fine. Only 1 out of 10 Fungi is good – the rest contain no insert or too many insert. And all the Archaea contain too long insert (~3000 bp). How is the possible? Does anyone have the same problem?
Be careful of UV exposure during gel isolation. Use long wave (365 nm) or blue light for illumination, and work quickly. Is the length of the bands for the archea and fungi correct on the gels?