After the vector and insert DNA have been prepared for ligation, determine the concentration either by spectrophotometer or by agarose gel against a known amount of standard marker. Test various vector:insert DNA ratios in order to find the optimum ratio for a particular vector and insert. In most cases either a 1:1 or a 1:3 molar ratio of vector:insert works well. The following example illustrates to conversion of molar ratios to mass ratios for a 3.0kb vector and a 500bp DNA insert: