Quantitative electroelution of oligonucleotides and large DNA fragments from gels and purification by electrodialysis.
We have designed a new device [Biotrap (Elutrap in the U.S.A. and Canada), available from Schleicher & Schuell] for electroelution, -concentration , and -dialysis of DNA and other charged macromolecules above 5 kDa. In an electric field, the DNA migrates in an open channel out of the gel slice through a microporous membrane, BT2, into the trap section, where it is retained by a very dense, non-adsorbant, and inert membrane BT1. Specifically designed for use in an electric field, the matrix of this new membrane is much denser than that of dialysis membranes. In contrast to dialysis membranes, BT1 will not adsorb large DNA fragments nor allow passage of small DNA fragments when subjected to an electric field. In the absence of an electric field, BT1 and BT2 effectively seal the trap, maintaining the final elution volume of the purified sample. The trap can contain from 200-600 microliter and is collected from above with a pipet. In the experiments described here, 85-95% of oligonucleotides (14-mer) and large (150 kb) DNA fragments were recovered, independent of fragment length. The purity of the eluted DNA was demonstrated by restriction enzyme digestion, nick-translation, primer extension, end-labeling with polynucleotide kinase, and ligation. Electrodialysis was successfully used for the complete removal of common contaminants inhibiting the polynucleotide kinase reaction and for the removal of CsCl from DNA samples.
Zentrum für Hygiene, Freiburg, F.R.G.
U
U
Göbel
R
R
Maas
A
A
Clad
D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D003956:Dialysis / Q000379:methods; D004563:Electrochemistry; D004586:Electrophoresis / Q000379:methods; D008567:Membranes, Artificial; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000302:isolation & purification*
D008567:Membranes, Artificial; D009838:Oligodeoxyribonucleotides; D004247:DNA