Picogram cloning and direct in situ sequencing of DNA from gel pieces.
We describe a simple and rapid procedure for cloning and sequencing of DNA fragments separated by gel electrophoresis, using novel hydrophilic gels, Clearose BG, Spreadex, and Poly(NAT), that do not melt at 95 degrees C. For cloning, a band of interest is excised precisely and incubated in an extraction buffer containing 5-10 mM MgCl2 at 70 degrees C 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.
Meijerink
E
E
Cytos Biotechnology AG, Schlieren, Switzerland.
Kozulic
B
B
Stranzinger
G
G
Neuenschwander
S
S
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D005647:Fucosyltransferases / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D013552:Swine; D000097631:Galactoside 2-alpha-L-fucosyltransferase
D004247:DNA; D017931:DNA Primers; D005647:Fucosyltransferases; D000097631:Galactoside 2-alpha-L-fucosyltransferase