Gentle alkaline denaturation
Highly stable DNA
The high-molecular-weight DNA generated through a REPLI-g Kit reaction should be regarded as high-quality genomic DNA and should be stored accordingly. For example REPLI-g amplified DNA can be stored for short periods at 4°C or –20°C for longer term storage. As with any high quality genomic DNA, to preserve the DNA quality, it should not undergo many freeze thaw cycles.
The stability of REPLI-g amplified DNA after long-term storage at -20°C was also investigated. Four different storage formats were used:
- 50 µl REPLI-g reaction without any further manipulation (“50 µl REPLI-g”)
- 50 µl REPLI-g reaction aliquoted to 5 µl volumes (“5 µl REPLI-g”)
- 50 µl REPLI-g reaction purified with QIAamp Mini Kit (“50 µl QIAamp purified REPLI-g”)
- 50 µl REPLI-g reaction diluted to a concentration of 50 ng/µl (“50 µl diluted REPLI-g”)
Amplified DNA (10 ng; as determined using a PicoGreen assay [for more information, refer to the REPLI-g Mini and Midi Handbook]) from each stored sample was analyzed at two different loci using real-time PCR (see figure Consistent long-term stability). No difference in the locus representation was detectable for the various storage conditions tested.