EZ1 ccfDNA Mini Kit (48)

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	Follow manufacturer’s instructions

Protocol tips
Follow manufacturer’s instructions

Publication protocol

Sample preparation and extraction.
Oligonucleotide fragments with sizes of 50, 100, 150, 200, 500, and 1,500 bp were constructed by the generation of random DNA sequences (Table 1) at GenScript (Piscataway, NJ). Imbedded within each oligonucleotide were identical forward and reverse primer sequences separated by random intervening sequences of different lengths for which a corresponding 6-carboxyfluorescein (FAM)-labeled black hole quencher (BHQ) probe was designed. Each fragment had an ATC at the 3′ end of the 5′ primer sequence and a TA at the 5′ end of the 3′ primer. Each of the fragments had a single amplicon sequence inserted into the fragment. In order to minimize PCR variability, the amplicons were designed to have very similar sizes; the amplicon sizes varied from 50 to 58 bp in length for the different fragment sizes. For each fragment size, the DNA fragments were assigned quantities on the basis of the results from the droplet digital PCR (ddPCR) assay and then diluted in EDTA plasma to concentrations of 4.0, 5.0, and 6.0 log10 copies/ml for subsequent extraction. The fragments were separately diluted in TE (Tris-EDTA) buffer for use as nonextracted controls. Aliquots of each fragment were frozen at −20°C and then shipped to the 3 participating laboratories. Four replicate extractions for each size/concentration were done with each of the 11 different instruments/methods, as described in Table 2. Kit selection and the method used for each assay were based on the manufacturers’ recommendations for optimum routine DNA virus extraction. All extractions were performed according to the manufacturers’ instructions. After extraction, the resulting eluates were frozen until tested with the ddPCR assay.

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Manufacturer protocol

Download the product protocol from Qiagen for EZ1 ccfDNA Mini Kit (48) below.

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