Efficient recovery of cloned human cytomegalovirus DNA fragments from agarose gels.
A simple and efficient method for the recovery of DNA fragments from agarose gels is described. After electrophoresis, bands of interest are cut out of the gel and agarose slices pushed through the opening of a syringe needle. The resulting gel slurry is frozen and thawed three times and then centrifuged. DNA in the supernatant is precipitated, resuspended in a small volume of buffer and, finally, desalted. A recombinant pAT153 plasmid carrying the BamHI-P fragment of the human cytomegalovirus (HCMV) genome was detected using purified viral HindIII-E DNA fragment as a probe. Restriction endonuclease analysis was used to confirm the identity of the cloned fragment. Experiments performed with the recombinant pLCR127 plasmid indicate that our freeze/thaw method, with about 80% recovery and very little DNA degradation, is more advantageous than the well known electroelution and NaI/glass methods.
Pramatarova
A
A
Centre de recherche en virologie, Institut Armand-Frappier, Laval-des-Rapides, Québec, Canada.
Yelle
J
J
D'Amours
B
B
Hamelin
C
C
D000327:Adsorption; D002498:Centrifugation; D011232:Chemical Precipitation; D003587:Cytomegalovirus / Q000235:genetics*; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000302:isolation & purification*; D004279:DNA, Viral / Q000302:isolation & purification*; D003956:Dialysis; D004587:Electrophoresis, Agar Gel; D005615:Freezing; D005898:Glass; D008863:Microspheres; D012974:Sodium Iodide
D004274:DNA, Recombinant; D004279:DNA, Viral; D004262:DNA Restriction Enzymes; D012974:Sodium Iodide