Animals were immediately washed in a Pipes homogenization buffer (0.25 mol [1.sup.-1] NaCl, 5 mmol [l.sup.-1] MgAc
, 25 mmol [1.sup.-1] EGTA, 1 mmol [1.sup.-1] DTT, 10 mmol [1.sup.-1] Pipes pH 6.8, 0.1% Triton X100) containing a protease inhibitor cocktail (catalog # P8340, Sigma Chemical Co.).
6 March 2014 - US provider of project and construction management services Mark G Anderson Consultants, or MGAC, said it had completed a merger with local cost and risk consultancy Basis LLC.
MGAC provides project and construction management services to corporations, government, hospitality and retail companies, as well as cultural and higher education institutions.
The purified DNA was digested with EcoRI and MseI restriction enzymes with digestion performed in a final volume of 25 [micro]L in 10 mM TRIS-HCl (pH 7.5), 10 mM MgAc
, 50 mM KAc, 5 mM DTT, 6 U EcoRI (Invitrogen Life Technologies, Carlsbad, CA), 4 U MseI (New England BioLabs, Inc., Beverly, MA), and 250 ng of genomic DNA for 2 h at 37[degrees]C, 15 rain at 70[degrees]C, and held at 4[degrees]C.
Briefly, 500 ng of wheat genomic DNA were subject to restriction-ligation in a single step during 6 h in a 30-[micro]L reaction mix (10 mM Tris-acetate pH 7.5, 10 mM MgAc
, 50 mM KAc, 5 mM DTT, 50 ng/mL BSA, PstI (5U), MseI (5U), and T4 DNA ligase 1U, 5 pmol PstI adaptors, 50 pmol MseI adaptors, and 12 pmol ATP).