Type-it Microsatellite PCR Kit (2000)

A multiplex PCR kit from QIAGEN developed and functionally validated for reliable microsatellite analysis. It is intended for endpoint PCR amplification of genomic DNA, with applications including multiplex PCR, microsatellites, STRs, VNTRs and SSRs. The kit is supplied as a mastermix formulation with hot-start capability.

On receipt, the kit should be stored at –30 to –15°C in a constant-temperature freezer. The 2x Multiplex PCR Master Mix contains 3 mM Mg2+ and HotStarTaq Plus DNA Polymerase, which requires a 5-minute heat-activation step at 95°C at the start of cycling. A 5x Q-Solution is included to support amplification of GC-rich or secondary-structure-rich templates by modifying DNA melting behaviour.

The protocol is optimized for fluorescent primers and downstream high-resolution fragment analysis on capillary sequencing instruments. Recommended parameters are 0.2 µM of each primer (with primer Tm ≥68°C), annealing for 90 s at 57–63°C (60°C is suitable for most systems), and a 3-step cycling program with extension at 72°C suited to targets up to 0.5 kb in length, followed by a 30 min final extension at 60°C. Starting from 50–200 ng template DNA per reaction, 20–24 cycles is recommended; 10–50 ng: 24–28 cycles; 0.1–10 ng: 28–32 cycles, with 28 cycles satisfactory for most cases and up to 200 ng template per 25 µl reaction. After cycling, samples are denatured for 5 min at 95°C, diluted 1:10 to 1:50 in deionized formamide or water together with a fluorescently labeled size standard, and then injected into a capillary sequencer.