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DNA Amplification Fingerprinting Protocol

DNA Amplification Fingerprinting Protocol

PCR amplification

The PCR reaction mix (10 µL) contains template DNA (2 ng/µL), primer (0.3 µM), Taq DNA polymerase, Stoffel Fragment (Perkin Elmer; 5 U), Mg++ (2.5 mM), buffer (1X) and overlaid with a drop of mineral oil. Amplifications are performed using the 96-well plates in a MJ Research thermal cycler for 35 cycles after an initial denaturation at 94C for 5 min and a final extension at 72C5 min. For arbitrary and mini-hairpin primers, each cycle consists of 5 sec at 94C, 20 sec at either 35 C or 45C (depending on the primer; see Table 2) and 30 sec at 72C For SSR primers, each cycle is 1 min at 94C, 1 min at 55C and 2 min at 72C.

Gel electrophoresis

DNA fragments are separated in a vertical electrophoresis system using a polyacrylamide-based vinyl polymer (GeneAmp; Perkin Elmer, Norwalk, CT); (He et al., 1994). Gels were prepared as follows:

Silver Staining for DNA visualization

Gels were silver stained using a modified procedure of Bassam et al. (1991) :

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