DpnI digest and purification

Following PCR amplification, residual (methylated) DNA template in each PCR reaction was DpnI digested at 37°C for 1 h. Each 110 μL digest reaction consisted of 95 μL PCR product, 11 μL 10x Fast Digest buffer, 1.5 μL Fast Digest DpnI (Thermo Fisher Scientific; Waltham, MA), and 2.5 μL deionized water. DpnI was inactivated at 80°C for 5 min, and DNA purification of each DpnI reaction was conducted with a PCR purification kit (Qiagen; Valencia, CA) according to the manufacturer’s protocol, each purified sample eluted with 50 μL of elution buffer.