Long PCR buffer

500 mM Tris-Cl (pH 9.0 at room temperature)

160 mM ammonium sulfate

1.5 mg/ml bovine serum albumin

25 mM MgCl₂

Sterilize the 10x buffer by filtration through an 0.22-μm membrane. Divide the sterile buffer into aliquots and store the aliquots at -20°C.

KCl (100 mM) can be used in place of ammonium sulfate in the 10x long PCR buffer, and gelatin can be used at a concentration of 0.01% in the final reaction in place of bovine serum albumin.

Additional components to improve the efficiency of long PCR include glycerol at a concentration of 5% (v/v) in the final reaction mixture to promote separation of DNA strands at lower temperatures, and EDTA at a concentration of 0.75 mM in the final reaction mixture to chelate divalent cations such as Mn²⁺ that might promote scission of DNA strands.