Appl Microbiol Biotechnol (2004) 65: 200–202 DOI 10.1007/s00253-004-1577-7 APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY R. M. Cranenburgh An equation for calculating the volumetric ratios required in a ligation reaction Received: 20 November 2003 / Revised: 15 January 2004 / Accepted: 16 January 2004 / Published online: 5 February 2004 # Springer-Verlag 2004 Abstract The ligation of two DNA fragments to create a new plasmid DNA molecule is a key reaction in molecular biology. Where the fragment lengths and concentrations are known, existing equations allow the desired relative molar ratio to be calculated, but this must then be related to the required volumes. Further calculations are then necessary if the maximum available volume is to consist of DNA solutions. The equation presented here allows the simple calculation of volumes of DNA solutions required to obtain a desired molar insert-to-vector ratio, and these can comprise all of the available volume in a ligation if required, thus maximising the yield of the recombinant plasmid. Introduction The ligation reaction is of fundamental importance to molecular biology by enabling the joining of vector and insert DNA fragments to create new plasmid DNA molecules. It is important to obtain the correct ratio of insert to vector for optimum recombinant plasmid gener- ation and to reduce the formation of concatomeric fragments, religated vectors, plasmid dimers and plasmids containing multiple inserts. A ligation reaction is usually carried out in a total volume of 10–20 μl at 4–37°C (often at room temperature) using the enzyme T4 DNA ligase, and a buffer containing Mg 2+ and ATP (typically from a stock concentration of ten times the working volume). T4 DNA ligase catalyses the joining of adjacent duplex DNA termini by the formation of phosphodiester bonds using ATP as a cofactor and can ligate either blunt or compatible cohesive ends. In an optimum ligation reaction aimed at inserting a single insert fragment into a vector, insert and vector DNA concentra- tions that disfavour monomolecular ring formation or concatamerisation should be used (Dugaiczyk et al. 1975; Sambrook et al. 1989). Existing ligation equations (e.g. Doyle and Miles 1996) only allow the molar ratios of insert and vector DNA to be calculated. As the length of a DNA molecule is directly proportional to its molecular weight, such equations offer no advantage over simply determining the ratio of fragment lengths. The equation described here allows the component volumes to be easily calculated given the DNA concentrations and fragment lengths, such that the chosen insert-to-vector molar ratio is achieved and the reaction can consist of DNA ligase, buffer, insert and vector DNA solutions only if required. Results Using the equation in Fig. 1, the volume of the vector component required in the ligation is calculated first, then this is subtracted from the total DNA component of the reaction (T) to give the required insert volume. The value T represents the combined volume of vector and insert DNA solutions added to the ligation reaction (e.g. T=8 μl in a 10-μl reaction with 1 μl each of ligase and buffer). The lengths and concentrations of the DNA fragments must be inserted along with the desired insert-to-vector ratio. The equation was tested to verify that the results generated for the predicted volumes did indeed give the correct molar ratios of DNA fragments (Table 1). Arbitrary values (input) were used to generate predicted volumes using the ligation equation (output). The moles of DNA were calculated to determine the insert-to-vector ratio in theoretical ligation reactions set up using the volumes generated by the equation (test). R. M. Cranenburgh (*) Cobra Biomanufacturing PLC, The Science Park, Keele, Staffordshire , ST5 5SP, UK e-mail: rocky.cranenburgh@cobrabio.com Tel.: +44-1782-714-181 Fax: +44-1782-799-817