NCJ Number
240011
Date Published
November 2007
Length
2 pages
Annotation
This protocol for forensic post-PCR purification using the MinElute PCR Purification Kit, which was adapted from Smith and Ballantyne (2007), is designed to purify double-stranded DNA fragments from PCR reactions resulting in high end-concentrations of DNA.
Abstract
Equipment and reagents to be supplied by the user are listed, and important steps before starting the protocol are described. The procedure is presented in nine steps. First, add five volumes of Buffer PB to one volume of the PCR sample and mix. Second, place a MinElute column in a 2-ml collection tube (provided). Third, to bind DNA, apply the sample to the MinElute column and centrifuge 1 minute; for maximum recovery, transfer all trace of the sample to the column. Fourth, discard flow-though and place the MinElute column back into the same tube. Fifth, in order to wash add 700 ml Buffer PE to the MinElute column and centrifuge 1 minute. Sixth, Discard flow-through and place the MinElute column back to the same tube. Seventh, repeat steps five and six three times to make a total of four washes. Eighth, discard flow-through and place the MinElute column back in the same tube; centrifuge the column for an additional 1 minute at maximum speed, and then centrifuge for 1 minute. For this step, it is advised that residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation. The ninth and final step is to elute DNA, add 10 ml Buffer EB (10 mM Tris-Cl, pH 8.5) to the center of the membrane, let the column stand for 1 minute, and then centrifuge for 1 minute. In this step, it is important to ensure that Buffer EB is dispensed directly onto the center of the membrane for complete elution of bound DNA.