Picogram cloning and direct in situ sequencing of DNA from gel pieces

摘要

We describe a simple and rapid procedure for cloning and sequencing of DNA fragments separated by gel electrophoresis, using novel hydrophilic gels, Clearose (R) BG, Spreadex (R), and Poly(NAT)(R), that do not melt at 95 degreesC. For cloning, a band of interest is excised precisely and incubated in an extraction buffer containing 5-10 mM MgCl2 at 70 degreesC for 15-45 min. The eluted DNA is added directly to the plasmid solution. Using a topoisomerase-based ligation system, we were able to transform bacteria with a few picograms of DNA and isolate recombinant clones. For in situ sequencing, the DNA in the gel serves as the template. No treatment before cycle sequencing is necessary for fragments up to 500 bp.