Application Guide - QIAxcel Advanced

110 QIAxcel Advanced Application Guide 10/2016 discarded and the pellet was used for DNA extraction with QIAamp DNA Stool Mini Kit. The MagAttract 96 cador Pathogen Kit can be used for high-throughput DNA extractions. One microliter of extracted DNA was used for each PCR reaction (data not shown). Multiplex PCR The primer mix was prepared in 1x TE buffer, and each primer had a final concentration of 0.11 µM. The PCR reaction mix (Table 2) was prepared by mixing 12.5 µl of 2x QIAGEN Multiplex PCR Master Mix, 2.0 µl of 25 mM MgCl2 , 8.5 µl RNase-free water and 1 µl template DNA (1–100 ng per 25 µl reaction; 10 ng DNA template is recommended). A modified PCR protocol described by Bai et al (4, 5) was used in this study. A 15 min denaturation step at 95° C was followed by a 35 cycles of amplification at 95° C for 20 s, 63° C for 30 s and 72° C for 90 s, with a final extension at 68° C for 10 min (Table 3). The following thermal cyclers have been tested and are compatible with this procedure and the described cycling conditions: TP600 thermal cycler (TaKaRa Bio Inc.), Eppendorf® Mastercycler® ep Gradient, Arktik Thermal cycler, Labnet PCR thermal cycler, Bio-Rad® C1000, Bio-Rad T 100 Thermal Cycler, MJ Research PTC-200, GeneAmp® PCR System 9700. QIAxcel Advanced protocol After amplification, PCR products were directly placed into the QIAxcel Advanced capillary electrophoresis system and separated using the QIAxcel DNA Screening Kit with the AM700 method, which includes the following electrophoresis parameters: alignment marker injection at Component Volume/reaction Final concentration RNase-free water 8.5 µl 2x QIAGEN Multiplex PCR Master Mix 12.5 µl 1x E. coli 11-plex Primer Mix 1.0 µl 0.11 µM 25 mM MgCl2 2.0 µl 5 mM* Total volume 24.0 µl Initial activation step 15 min 95°C 3-step cycling: Denaturation 20 s 95°C Annealing 30 s 63°C Extension 90 s 72°C Number of cycles 35 Final extension 10 min 68°C Hold 8°C Table 2. Preparation of reaction mix. Table 3. PCR cycling conditions. * Final concentration includes MgCl2 in the QIAGEN Multiplex PCR Master Mix

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