HpyCH4III NEB#R0618

Protocol tips

Upstream tips	Protocol tips	Downstream tips
For some samples, it was necessary to modify the amount of DNA in the PCR, PCR product in the second reaction or PCR product used in the digestion.	Following amplification (verified by agarose gel electrophoresis), PCR products were digested with the restriction enzyme HpyCH4III (New England biolabs, Ipswich, MA, USA). The restriction enzyme profile was identified using NEBcutter33. The digestion was performed in 10 μL containing 0.5 μL of the enzyme (5U/μL), 1 μL of the enzyme’s buffer and 5 μL of PCR product (2–7 μL according to the intensity of PCR products on agarose gels). The digestion was incubated at 37 °C for 3 hours.	All digestion reaction and the equivalent amount of non-digested DNA were visualized in electrophoresis on 3% agarose gel and examined under a UV transilluminator.

Upstream tips
For some samples, it was necessary to modify the amount of DNA in the PCR, PCR product in the second reaction or PCR product used in the digestion.
Protocol tips
Following amplification (verified by agarose gel electrophoresis), PCR products were digested with the restriction enzyme HpyCH4III (New England biolabs, Ipswich, MA, USA). The restriction enzyme profile was identified using NEBcutter33. The digestion was performed in 10 μL containing 0.5 μL of the enzyme (5U/μL), 1 μL of the enzyme’s buffer and 5 μL of PCR product (2–7 μL according to the intensity of PCR products on agarose gels). The digestion was incubated at 37 °C for 3 hours.
Downstream tips
All digestion reaction and the equivalent amount of non-digested DNA were visualized in electrophoresis on 3% agarose gel and examined under a UV transilluminator.

Publication protocol

Following amplification (verified by agarose gel electrophoresis), PCR products were digested with the restriction enzyme HpyCH4III (New England biolabs, Ipswich, MA, USA). The restriction enzyme profile was identified using NEBcutter33. The digestion was performed in 10 μL containing 0.5 μL of the enzyme (5U/μL), 1 μL of the enzyme’s buffer and 5 μL of PCR product (2–7 μL according to the intensity of PCR products on agarose gels). The digestion was incubated at 37 °C for 3 hours. All digestion reaction and the equivalent amount of non-digested DNA were visualized in electrophoresis on 3% agarose gel and examined under a UV transilluminator. Electrophoresis was performed in a room specific for amplified DNA, with appropriated sets of pipettes and plugged pipettes tips. For some samples, it was necessary to modify the amount of DNA in the PCR, PCR product in the second reaction or PCR product used in the digestion.

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