DpnI NEB#R0176

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	The PCR reaction was followed by an overnight digestion at 37°C through addition of 2 μl (4000 units) of DpnI (NEB, Hitchin, UK). 5 μl of the PCR reaction were subsequently transformed in E. coli strain Top10. Ten colonies per plate were isolated and analysed via restriction digest and sequencing (GATC Biotech, Konstanz, Germany).

Protocol tips
The PCR reaction was followed by an overnight digestion at 37°C through addition of 2 μl (4000 units) of DpnI (NEB, Hitchin, UK). 5 μl of the PCR reaction were subsequently transformed in E. coli strain Top10. Ten colonies per plate were isolated and analysed via restriction digest and sequencing (GATC Biotech, Konstanz, Germany).

Publication protocol

The SDM reaction was set up in two individual reactions one for each primer (0.3 μM final concentration). The reactions were set up in KOD Hotstart buffer and a final concentration of 0.2 mM each dNTP, 2.25 mM Mg2+, 5% DMSO, 20 ng template DNA and one unit of KOD Hotstart Polymerase (Merck, Darmstadt, Germany). An initial 95°C denaturation for two minutes was followed by cycles of 30 seconds denaturation at 95°C, one minute annealing at 55°C and synthesis of six minutes (one minute per kbp) at 70°C. After twelve cycles the two individual reactions were pooled and subjected to another 15 cycles of 30 seconds of denaturation at 95°C and seven minutes of synthesis at 70°C omitting an individual annealing step to increase specificity of primer template binding. The PCR reaction was followed by an overnight digestion at 37°C through addition of 2 μl (4000 units) of DpnI (NEB, Hitchin, UK). 5 μl of the PCR reaction were subsequently transformed in E. coli strain Top10. Ten colonies per plate were isolated and analysed via restriction digest and sequencing (GATC Biotech, Konstanz, Germany).

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