HapII (HpaII, MspI) restriction enzyme

Protocol tips

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	20 units EcoRI (Takara, P.R.China) and 20 units HpaII (Takara, P.R.China) were used to digest 200 ng genomic DNA in 20 µl of the reaction mixture at 37°C for 2 h.

Protocol tips
20 units EcoRI (Takara, P.R.China) and 20 units HpaII (Takara, P.R.China) were used to digest 200 ng genomic DNA in 20 µl of the reaction mixture at 37°C for 2 h.

Publication protocol

"MSAP method was mainly described as follows: at first, genomic
DNA of cotton was divided into two groups, for the first group, 20
units EcoRI (Takara, P.R.China) and 20 units HpaII (Takara,
P.R.China) were used to digest 200 ng genomic DNA in 20 μl of
reaction mixture at 37°C for 2 h. For the second group, instead of
HpaII, the MspI (Takara, P.R. China) was used in combination with
EcoRI to digest 200 ng genomic DNA with the same condition. The
enzyme digestion reaction was terminated by incubation at 65°C for
10 min. Then, the ligation reaction was proceeded in a final volume
of 40 μl mixture, which contains 1 unit T4 DNA ligase, 0.2 mM ATP,
5 pm EcoRI adapters and 50 pm HpaII/MspI adapters for additional
6 h at 20°C (Table 1). Subsequently, the ligation mixture was usedas the template for the preselected amplification reaction with
EcoRI+A and HpaII / MspI+T primers (Table 1). The polymerase
chain reactions were carried out in a 25 μl reaction mixture with 1 μl
of ligation reaction mixture, 50 ng of E+A primer, 50 ng of HM +T
primer, 0.5 unit Taq DNA polymerase (Biocentury transgene, P.R.
China), 0.2 mM dNTP (Biocentury transgene, P.R.China) and 2.5 μl
of 10× polymerase buffer (Biocentury transgene, P.R. China) for 21
cycles with 1 min denaturation at 94°C, 1 min annealing at 56°C,
and 1 min extension at 72°C. The preselected amplification
products were used as the template for the selective amplification
reaction. The primers were the EcoRI and HpaII/MspI primers with
two added selective nucleotides (Table 1). The polymerase chain
reactions were carried out in total volumes of 25 μl, containing 0.3
μl of pre-amplification product, 50 ng of EcoRI primer, 50 ng of
HpaII/MspI primer, 1 unit Taq polymerase, 0.5 mM dNTP and 2.5 μl
of 10× PCR buffer. The PCR procedure was performed according to
the standard amplified fragment polymorphism touchdown protocol
(Vos et al., 1995). The products of selective amplification were
mixed with 8 μl of denaturating buffer (98% formamide, 10 mM
EDTA, 0.1% bromphenol blue, and 0.1% xylene cyanol), then
denatured at 95°C for 5 min and separated on 6% polyacrylamide
gel in 1× 44.5 Mm Tris/Borate, 0.5 Mm EDTA, pH 8.0 (TBE) buffer
at 80 watts for 1 h. Gels were stained according to the silver
staining method described by Bassam et al. (1991)."

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