CloneChecker™ System (for screening bacterial colonies)

Depending on the copy number of the plasmid, the number of cells collected, and their stage of growth, it is estimated that ~2 to 20 ng are obtained for analysis. This amount is suitable for one gel analysis. Typically, the Supercoiled Analysis method recovers more plasmid than the Restriction Enzyme Analysis method. Please note, plasmid is not purified with this system, but rather only released from cells in manner suitable for characterization. Plasmids remain contaminated with most of the cell constituents.

Restriction Enzyme Analysis and Supercoiled Analysis are the methods provided in the CloneChecker system. Restriction Enzyme Analysis is useful for positively identifying clones from a characteristic digestion pattern. Insert size and orientation can be determined reasonably accurately by choosing the appropriate restriction endonuclease(s) and an appropriate DNA molecular size marker. This screening procedure makes sense when the probability of finding the correct clone within the population of colonies on a plate is reasonably high, or when confirmation of a clone resulting from a preliminary screening is desired. Supercoiled Analysis is useful when the recovery frequency of an insert-containing vector is low or when the desired insert is within a certain size range. Within a few minutes of picking a clone, a sample can be loaded on an agarose gel for determination of supercoiled size without the extra time or expense of performing a restriction digest on the plasmids. Both methods avoid overnight culture of each clone and the work plasmid DNA minipreps for this type of analysis.

The system contains sets of proprietary chemical solutions that release nucleic acids, especially plasmids, from bacterial cells. Typically, released plasmids are then characterized physically by supercoiled size or by restriction digestion pattern using agarose gel electrophoresis.

Plasmids ranging in size from 2.7 kb to 13 kb have been analyzed, and the CloneChecker System has been used to isolate a 46 kb cosmid for quick analysis. It should be possible to assay for larger DNAs, such as cosmids, providing a sufficient amount of DNA is detectable.

Restriction endonuclease digestion of released plasmid DNA was successful with 20 of the popular enzymes used in cloning: Acc I, Ava I, BamH I, BstE II, Bgl II, Cla I, EcoR I, Hpa I, Hind III, Kpn I, Nco I, Nhe I, Not I, Sac II, Sal I, Ssp I, Sst I, Spe I, Xba I, and Xho I. In addition to single digestions with these enzymes, a number of double digests were successful as well. No enzyme tested has failed to perform properly. Note, restriction digests are only possible with plasmid released with the GREEN solution from the Restriction Enzyme Digest method.