Straightforward Procedure for Laboratory Production of DNA Ladder

DNA ladder is commonly used to determine the size of DNA fragments by electrophoresis in routine molecular biology laboratories. In this study, we report a new procedure to prepare a DNA ladder that consists of 10 fragments from 100 to 1000？bp. This protocol is a combination of routinely employed methods: cloning, PCR, and partial digestion with restriction enzymes. DNA fragments of 100？bp with unique restriction site at both ends were self-ligated to create a tandem repeat. Once being cloned, the tandem repeat was rapidly amplified by PCR and partially digested by restriction enzymes to produce a ladder containing multimers of the repeated DNA fragments. Our procedure for production of DNA ladder could be simple, time saving, and inexpensive in comparison with current ones widely used in most laboratories. 1. Introduction A DNA ladder is a solution of DNA molecules of different lengths used in agarose or acrylamide gel electrophoresis. It is applied as a reference to estimate the size of unknown DNA molecules that were separated based on their mobility in an electrical field through the gel. Thus, DNA ladders are essential molecules routinely used in every DNA dealing laboratory. Digestion with restriction enzymes of known-length DNA fragments from natural sources such as lambda [1], simian virus 40 [2], and plasmid [3] creates the ladder fragments the lengths of which are dependent on restriction enzyme sites thus, not fully controlled. To overcome this disadvantage and to make DNA ladders more flexible, DNA engineering was developed [4]. Perhaps, for commercial purpose, DNA manipulation for producing DNA ladder fragments became confidential. Typically, a DNA fragment that contains a tandem repeat units separated by the same unique restriction enzyme sites was cloned into a plasmid and then partially digested to produce a ladder with multimers of the repeats [5, 6]. However, the more clear visibility of small size fragments requires the higher amount of plasmid used in cleavage reaction. Recently, many laboratory protocols describing the preparation of DNA ladders by employing the polymerase chain reaction (PCR) method have been reported [7–9]. This method involves either the simultaneous amplification of a DNA target using primer sets [8] or the separate amplification of a different DNA targets using specific primers [9, 10]. However, using simultaneous primer sets is often difficult to be reproductive because of problematic optimization of PCR conditions, while using separate primer set for a particular fragment of ladders causes a laborious task.