Marked improvement of PAC and BAC cloning is achieved using electroelution of pulsed-field gel-separated partial digests of genomic DNA.
We describe a simple electroelution method for purifying large, gel-fractionated DNA molecules that alleviates the need for melting of the agarose and subsequent enzymatic agarose digestion. The method yields DNA that is visibly more intact than that purified from a standard agarose-digestion protocol and is more amenable to large-fragment cloning with PAC and BAC vectors. These findings are notable in that PAC and BAC library construction is a very labor-intensive and costly procedure, such that any net improvement in cloning efficiency is highly advantageous. This method also should prove useful towards other applications which require purification of very large DNA molecules, such as YAC cloning.
Strong
S J
SJ
Department of Pediatrics, University of South Florida, All Children's Hospital, 801 Sixth Street South, St Petersburg, FL 33701, USA.
Ohta
Y
Y
Litman
G W
GW
Amemiya
C T
CT
D000818:Animals; D002875:Chromosomes / Q000235:genetics*; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics; D002876:Chromosomes, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D005822:Genetic Vectors; D016678:Genome; D012685:Sepharose; D012860:Skates, Fish / Q000235:genetics
D004247:DNA; D012685:Sepharose