Construction of Large-Insert BAC Library from C57BL/6J Mouse
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We describe the newest version of the protocol toconstruct a bacterial artificial chromosome (BAC)library which carries uniform and larger insertDNA. This protocol is based on the five majorimprovements reported by Osoegawa et al. (1998)to increase the cloning efficiency and decrease thelevel of non-insert clones. The first improvement isa purification of high molecular weight (HMW)DNA with a pulse field gel electrophoresis, whicheliminates inhibitory contaminants for restrictionenzymes and leads to the increase of digestionefficiency of HMW DNA. The second is the doublesizefractionation method, in which partially digestedDNA is subjected to an electrophoresis in reversedirection to remove smaller DNA fragments, andthen size-fractionated in forward direction. This stepenables the preparation of uniform and large-sizeinsert DNA without contamination by smaller DNA.The third is recovering the size-fractionated DNAfrom agarose gel by the use of electroelution, ratherthan the use of β agarase as in conventional methods.This step dramatically increases both the purity andintegrity of large DNA fragments, leading to anincrease of cloning efficiency. The fourth step is theconcentration of the ligation products using dialysisagainst polyethylene glycol buffer with high osmoticpressure. This step increases the number of thetransformants produced in a single transformationtrial, resulting in saving time and cost. The fifth isthe most effective improvement in preparing a BACvector to minimize a level of non-insert clones. Thisstep employs the ligase-treatment of vector fractionto completely remove the molecules with phosphorylatedends.<br>In this paper, we introduce the precise optimalconditions in each step, which enabled us to makeBAC libraries with around 200 kb insert DNA. Oneexample is the RPCI23 library constructed from C57BL/6J female mice. This library consists ofapproximately 180,000 clones with an averageinsert size of 197 kb and contains only 6.8 % noninsertclones. This library covers the mouse genome11.2 times with the most uniform and largest insertDNA except for YAC libraries.
bioimages 6(3), 117-125, 1998-12-01