Generation of Control Ligation Product Libraries for 3C Analyses

Control libraries are used to determine differences in the efficiency of polymerase chain reaction (PCR) primers in detecting 3C ligation products as well as any differences in the efficiency of amplification. Control libraries are generated by random ligation of restriction fragments so that each ligation product is present in equimolar amounts. This protocol begins with preparations of genomic DNA or BAC DNA preparations. The restriction enzyme used here should be the same as the one used for generation of the 3C or ChIP-loop library. For small genomes, such as those of bacteria and yeast, this protocol can be started with purified genomic DNA. For organisms with larger genomes, such as mouse and human, control libraries are generated from purified BAC clones that cover the genomic region under study. When the region of interest is large and multiple BAC clones are required, select BAC clones that overlap only minimally. Any gaps between the regions covered by the BACs should also be kept to a minimum. Several kits for purifying BAC clones are available (e.g., the Large-Construct Kit from QIAGEN). BAC clones should be mixed in equimolar amounts to ensure that each restriction fragment is present in equal concentration. This is achieved by quantifying the molar concentration of each BAC preparation using real-time PCR and primers that recognize the common BAC vector backbone. Alternatively, the concentration of BAC preparations can be quantified using gel quantification or a NanoDrop detection system.