HpaII NEB#R0171

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	DNA (1 μg) was digested in NEB buffer 1 with 10 U of HpaII (NEB) in a 20-μl reaction volume for 3 hours at 37°C.

Protocol tips
DNA (1 μg) was digested in NEB buffer 1 with 10 U of HpaII (NEB) in a 20-μl reaction volume for 3 hours at 37°C.

Publication protocol

The MSAP procedure was performed in two technical replicates starting from the same DNA sample. DNA (1 μg) was digested in NEB buffer 1 with 10 U of HpaII (NEB) in a 20-μl reaction volume for 3 hours at 37°C. Ten microliters of the reaction mixture was transferred to 10 μl of NEB 2 buffer with 5 U of MspI (NEB) and incubated for 3 hours at 37°C. Simultaneously, 0.5 μg of the same original DNA sample was incubated in NEB 2 buffer with 5 U of MspI (NEB) in a 10-μl reaction volume for 3 hours at 37°C. Next, 10 μl of EcoRI buffer (NEB) with 5 U of EcoRI (NEB) were added to 10-μl reactions (HpaII and MspI digestions) and 5 μl of 2.5× EcoRI buffer (NEB) with 5 U of EcoRI (NEB) were added to a 20-μl HpaII + MspI reaction volume followed by incubation for an additional 1.5 hours at 37°C. EcoRI_ADAPTER1(F) 5′-CTCGTAGACTGCGTACC-3′ (10 μM)/ EcoRI_ADAPTER2(R) 5′-AATTGGTACGCAGTCTAC-3′ (10 μM) and HpaII/MspI_ADAPTER1(F) 5′-GATCATGAGTCCTGCT-3′ (100 μM)/ HpaII/MspI_ADAPTER2(R) 5′-CGAGCAGGACTCATGA-3′ (100 μM) were used to prepare 5 μM of EcoRI and 50 μM of MspI/HpaII adapters, respectively, by mixing appropriate oligonucleotides and incubating at 98°C for 5 min followed by slow cooling in a polystyrene box (for approximately 6 hours). Six microliters of digested DNA samples was added to 3.75 μl of mix comprising 1 μl of 5 μM EcoRI adapter (5 pmol), 1 μl of 50 μM MspI/HpaII adapter (50 pmol), 1 μl of fresh 10× T4 DNA ligase buffer, and 0.75 μl of sterile redistilled water. After mixing and heating to 37°C, 0.25 μl of T4 DNA ligase (100 U, NEB) was added, and the mix was incubated at 37°C for 3 hours followed by incubation at 20°C overnight. Non-ligated adapters were removed, and ligated DNA was purified using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel). DNAs were eluted in 2 × 10 μl of 5 mM Tris-Cl (pH 8), and 3 μl was used in 10-μl preselective amplification reactions with EcoRI_A 5′-GACTGCGTACCAATTCA-3′ and HpaII/MspI_T 5′-ATGAGTCCTGCTCGGT-3′ primers at 200 nM concentration each. In addition, the preselective reactions contain DynazymeII buffer, 125 μM dNTPs, and 0.5 U of DynazymeII DNA Polymerase (Thermo Scientific). The conditions of the preselective amplification were as follows: 72°C for 5 min, 94°C for 3 min followed by 30 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 2 min. A final step at 60°C for 10 min was also added. Five microliters of preselective products was checked by electrophoresis in 2% agarose gels (visible as a smear from 100 to 1000 bp) and the remaining 5 μl was diluted by adding 50 μl of redistilled water. The diluted preselective product (2.5 μl) was amplified using fluorescently labeled EcoRI_ACT[HEX] (green peaks in Figure 3 and Additional file 1: Figure S1) and labeled HpaII/MspI_TTC[6-FAM] (blue peaks in Figure 3) or HpaII/MspI_TAG (Additional file 1: Figure S1) selective primers (200 nM each) in a 12.5-μl total reaction volume containing KAPA buffer B, 125 μM dNTPs, and 0.5 U of KAPA Taq DNA Polymerase (KAPA Biosystems). The conditions of the selective amplification were as follows: 94°C for 3 min, 13 cycles of 94°C for 30 s, 65°C for 30 s reduced by 0.7°C per cycle, and 72°C for 2 min followed by 24 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 2 min. A final step at 72°C for 10 min was also added. The product of selective amplification (5 μl) was checked by 2% agarose gel electrophoresis. Next, 1.5 μl of the selective amplification product was added to a mix of 10 μl of deionized formamide for capillary electrophoresis (Genomac) and 0.3 μl of GMC GT500-L DNA standard (Genomac) that is fluorescently labeled with LIZ dye (orange peaks in Figure 3 and Additional file 1: Figure S1). DNA was denaturated by heating at 98°C for 3 min followed by cooling on ice. Denatured DNA was separated using the fragment analysis option in the ABI Prism 3100 Genetic Analyzer. The obtained electropherograms were aligned according the signal of the DNA standard, analyzed and visualized using GeneMarker version 1.80 (SoftGenetics).

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