Genotyping by polymerase chain reaction (PCR) is an important technique for screening mutants in various organisms [1
] and genome editing by CRISPR/Cas9 [4
]. Mutant screening by PCR is efficient for multiple samples. Escherichia coli
colony-PCR is also an efficient technique for screening positive colonies [8
], especially for high-throughput-cloned E. coli
plasmids expressing recombinant proteins [11
]. Microfluidic devices have recently been coupled with PCR as a method for high-throughput PCR screening [12
]. However, conventional PCR using plasticware (0.2-mL 96-well PCR plates) are also widely used in high-throughput PCR screening. Since screening reactions include small volumes of PCR mixtures (~10 µL), the solution is required to be spun down before agarose gel electrophoresis [13
]. Several commercially available electric centrifuges can be used for this purpose. However, these centrifuges require a few seconds for acceleration and deceleration to spin the PCR solutions down in 96-well PCR plates. Moreover, the machines require electricity and occupy significant amounts of space on laboratory benches.
A commercially available salad spinner can be used as a low-cost alternative to cytocentrifuges [16
]. The basket in the salad spinner can rotate at ~600 revolutions per minute (rpm) [16
]; this speed of rotation and centrifugal force is sufficient for hematocyte preparations [16
]. Recently, a high-speed hand-powered paper centrifuge was developed for separation of plasma from whole blood [18
]. The paper centrifuge can simultaneously process eight samples with high centrifugal forces of 30,000× g
. In this study, I developed a manual centrifuge to spin solutions down in 96-well PCR plates for conventional PCR screening, thereby significantly improving the commercially available salad spinner. The manual centrifuge is lightweight, portable, and usable in any space.
4. Expected Results
PCR solutions in 96-well PCR plates were sufficiently centrifuged using the manual centrifuge for 3 s (Figure 3
A). DNA fragments from these centrifuged PCR mixtures could be clearly visualized by DNA agarose gel electrophoresis (Figure 4
In contrast, using the conventional electric centrifuge resulted in poor centrifugation after 3 and 10 s (Figure 3
B,C); this could be attributed to the long time required for acceleration and deceleration. Centrifugation for 20 s by the electric centrifuge was necessary to sufficiently spin the 96-well PCR plates (Figure 3
D). In contrast to the manual centrifuge, the electric centrifuge has a heavy metal drive system for centrifugal forces higher than 500× g
, which is associated with a longer duration of acceleration and deceleration during centrifugation. The manual centrifuge developed in this study is a light plasticware that can accelerate and decelerate quickly to help the mixtures with settling down. Therefore, although the manual centrifuge did not rotate at a high speed or centrifugal force, 3 s was enough to spin the solutions down in the 96-well PCR plates. The manual centrifuge using a commercially available salad spinner was previously developed with a relative centrifugal force of 24× g
for centrifugation of blood samples (Table 1
]. The manual centrifuge developed in this study was modified to centrifuge solutions in 96-well PCR plates and its centrifuge force (25× g
) was similar to that previously reported [16
]. Although the manual centrifuge developed in this study has to be constructed by the user, its construction is simple and takes a short time (~30 min). The polystyrene foam base was simply cut out and set into the bottom of the salad spinner to fit two 96-well PCR plates.
shows the features of the two centrifuges for the 96-well PCR plates. The conventional electric centrifuge for 96-well PCR plates had insufficient acceleration and deceleration and required a few tens of seconds to spin the solutions down in the 96-well PCR plates. Moreover, the electric centrifuge had a built-in electric motor, which made it heavy owing to the presence of multiple components. Therefore, the machine was hard to move and constantly occupied a significant amount of space on the laboratory bench. In contrast, the manual machine developed in this study was light (~0.59 kg) and could be carried with one hand. When not needed, the machine could be stowed under a laboratory bench or on a shelf. Recent research utilizes a wide range of techniques from molecular biology to biochemistry. Thus, the efficient use of small laboratory space for successfully performing a variety of experiments requires equipment to be easily portable.
The manual centrifuge was associated with quick acceleration and deceleration and sufficiently spun the solutions in the two 96-well PCR plates after 3 s. Operation of the manual centrifuge did not require electricity, and the instrument functioned on a simple mechanism, and its operation was effortless. Moreover, the price of the salad spinner for the body of the manual centrifuge was notably less expensive than an electric centrifuge, costing only ~USD 18 (i.e., 1/40th of the cost of one electric centrifuge). Thus, the manual centrifuge developed in this study was simple, fast, inexpensive, and useful for spinning solutions down in 96-well PCR plates.