The surface was then washed with TE (10 mM Tris HCl, 1 mM EDTA, pH8.0) twice, equilibrated with digestion buffer (NEB buffer 3), then incubated with the restriction endonuclease SwaI New Englandd Biolabs, Beverly, MA), which cleaves the genomic DNA at its cognate site.
After washing, the plugs were sliced and then digested in fresh restriction buffer with the enzyme SmaI (50 U/ µL) at 25°C for 12 h.
The inserts of the first 96 clones were digested with the restriction enzymes BsuR1 (HaeIII) and AluI (Fermentas) overnight at 37°C with the appropriate buffer and 10 units of enzyme.
PCR-RFLP with the restriction enzyme ApeK1 was used to detect the mutation according to the manufacturer's recommendations for time, temperature and buffer conditions.
After thorough washing in TE buffer, the plug was subjected to digestion with either of the restriction endonucleases AscI, ApaI, I-CeuI, or SmaI.
After a minimum of 2 hours equilibration with the respective restriction enzyme buffer, blocks were incubated with restriction enzymes for 48 hours at 37°C.
Forested buffer strips with restrictions on timber harvest and road-building (i.e. riparian buffer zones) were originally implemented to protect aquatic functions and resources.
The solution was then diluted 2-fold with the same restriction buffer, and Triton X-100 was added to a final concentration of 1.8% (to sequester the SDS) followed by incubation at the same conditions for another hour.
About 200 ng of genomic DNA from each sample was double digested separately with 5 U of the restriction enzymes SacI and MseI (Fermentas) with 1 × restriction buffer in a reaction volume of 25 μl.
The genomic DNA was digested with 50 units of the restriction enzymes BssHII in 250 μl of 1X buffer 3 for 5 hours at 37°C.
