REPLI-g Mini Kit (100)

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	Implementation of modifications for developing optimized conditions

Protocol tips
Implementation of modifications for developing optimized conditions

Publication protocol

Whole-genome amplification: All non-MDA WGA were performed following individual kit manufacturer's instructions. MDA-based WGA was performed using either REPLI-g Mini kit (Qiagen) or Genomiphi kit (GE Healthcare). For Genomiphi, the kit manufacturer's instructions were followed without modification. For the REPLI-g Mini kit, manufacturer's instructions were followed during preliminary tests. The following modifications were performed in developing optimized conditions for the REPLI-g Mini kit: nuclease-free water and all tubes were UV-treated before use. WGA reactions were performed in 0.2 ml PCR tubes. Buffer D1 stock solution (Qiagen) was reconstituted by adding 500 µl of nuclease-free water, and a working solution was prepared by mixing the stock solution and nuclease-free water in the ratio of 1 : 3.5, respectively. Unmodified Buffer N1 was reconstituted by mixing Stop solution (Qiagen) and nuclease-free water in the ratio of 1 : 5.7. Modified buffer N1 was prepared by including tetramethylammonium chloride (TMAC) at a concentration of 300 mM. To denature DNA templates, 5 µl of the DNA solution was mixed with 5 µl of buffer D1 (working solution prepared as described above). The mixture was vortexed and centrifuged briefly before incubating at room temperature for 3 min. Denatured DNA was neutralized by adding 10 µl of either unmodified or modified buffer N1. Neutralized DNA was mixed by vortexing and centrifuged briefly. To amplify the DNA template, denatured and neutralized sample was mixed with 29 µl of REPLI-g Mini Reaction Buffer and 1 µl of REPLI-g Mini DNA polymerase to obtain a final reaction volume of 50 µl. The reaction mixture was incubated at 30°C for 16 h using an MJ Research PTC-225 thermal cycling system (GMI, Inc., USA) with the heating lid set to track at +5°C. Amplified DNA was cleaned using Agencourt Ampure XP beads (Beckman Coulter) using sample to beads ratio of 1 : 1 and eluted with 50 µl of EB (Qiagen).



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Manufacturer protocol

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