Isolation and purification of large DNA restriction fragments from agarose gels.
This unit describes methods for recovering and purifying DNA restriction fragments from agarose gels. The first protocol describes electroelution of the fragment of interest from standard agarose gels using buffer-filled dialysis bags, followed by concentration and purification using an Elutip column. This approach can be used effectively for fragments of all sizes from 50 to 20,000 bp. Electrophoresis directly onto NA-45 paper (second protocol) provides relatively high yields for fragments <2000 bp. Fragments >1000 bp can also be separated on low gelling/melting agarose gels and purified by phenol extraction (third protocol), b-agarase digestion of the gel (first alternate protocol), or via glass beads extraction (second alternate protocol). Removing linkers from a fragment using a column rather than a gel is described, followed by a method for estimating DNA concentrations in solution.
Moore
David
D
Baylor College of Medicine, Houston, Texas, USA.
Dowhan
Dennis
D
Chory
Joanne
J
Ribaudo
Randall K
RK
D000180:Acrylic Resins; D002853:Chromatography, Liquid / Q000379:methods; D002854:Chromatography, Paper / Q000379:methods; D004247:DNA / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D004996:Ethidium; D007202:Indicators and Reagents; D012150:Polymorphism, Restriction Fragment Length; D012822:Silicon Dioxide; D013696:Temperature
D000180:Acrylic Resins; D007202:Indicators and Reagents; C037239:Sephacryl Superfine; D012822:Silicon Dioxide; D004247:DNA; D004996:Ethidium