HhaI restriction enzyme

Protocol tips

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	Genomic DNA (500 ng) was digested with 50 U of HpaII, HhaI, MspI (TaKaRa), or McrBC (New England Biolabs) overnight at 37°C in 50 μl of the recommended buffers. The digested DNA was recovered by standard ethanol precipitation and dissolved in 10 μl of TE (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). Note that the amount of DNA used for digestion is dependent on the number of amplicons to be tested. For smaller amount of DNA, use of appropriate carriers would be helpful to ensure efficient recovery.

Protocol tips

Genomic DNA (500 ng) was digested with 50 U of HpaII, HhaI, MspI (TaKaRa), or McrBC (New England Biolabs) overnight at 37°C in 50 μl of the recommended buffers. The digested DNA was recovered by standard ethanol precipitation and dissolved in 10 μl of TE (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). Note that the amount of DNA used for digestion is dependent on the number of amplicons to be tested. For smaller amount of DNA, use of appropriate carriers would be helpful to ensure efficient recovery.

Publication protocol

"Genomic DNA (500 ng) was digested with 50 U of HpaII, HhaI, MspI (TaKaRa), or McrBC (New England Biolabs) overnight at 37°C in 50 μl of the recommended buffers. The digested DNA was recovered by standard ethanol precipitation and dissolved in 10 μl of TE (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). Note that the amount of DNA used for digestion is dependent on the number of amplicons to be tested. For smaller amount of DNA, use of appropriate carriers would be helpful to ensure efficient recovery.

The DNA digested with each enzyme was then amplified using GenomiPhi Amplification Kit (GE Healthcare). We routinely use 50 ng of digested DNA for each amplification. Briefly, 1 μl (50 ng) of digested DNA prepared as above was mixed with 9 μl of sample buffer and then denatured for 5 min at 96°C. Following cooling down to 4°C, the denatured DNA was mixed with 9 μl of reaction buffer and 1 μl of phi29 DNA polymerase, and then incubated for two days at 30°C. For inactivation of phi29 DNA polymerase, the amplified DNA solution was heated for 10 min at 65°C. The amplified DNA was purified by ethanol precipitation and dissolved in 100 μl of TE. Here, ~5 μg of whole-genome-amplified DNA was obtained from 50 ng of digested DNA.

We used 1 μl (50 ng) of each WGA product prepared as above for each PCR in 10 μl of the recommended buffers containing 2.5 U of Ex-Taq DNA polymerase (TaKaRa) and 2.5 pmols of each primer. Since each HM-PCR assay involves 4 PCR, HM-WGA-PCR uses 200 ng (= 50 ng × 4) of amplified DNA in total, which is equivalent to 2 ng of original genomic DNA. Thermal cycling parameters and primer sequences used in this study are shown in Additional file 1: Supplemental Table S1."

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Manufacturer protocol

Download the product protocol from Takara Bio Inc for HhaI restriction enzyme below.

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