A Simple and Fast Manual Centrifuge to Spin Solutions in 96-Well PCR Plates

A simple and fast manual centrifuge was developed to spin down solutions in 96-well polymerase chain reaction (PCR) plates. A commercially available salad spinner was utilized for this purpose. Acceleration and deceleration of the centrifuge were faster than those of a conventional electric centrifuge using 96-well PCR plates. Solutions in a 96-well PCR plate settled quickly after centrifuging for only 3 s. This lightweight centrifuge can be stored under a laboratory bench or on a shelf and can be put on the bench only when required, whereas the electric centrifuge is immobile due to its weight and the requirement of electric cables. This simple centrifuge is inexpensive, requires minimal effort for making, and can be used anywhere.


Introduction
Genotyping by polymerase chain reaction (PCR) is an important technique for screening mutants in various organisms [1][2][3] and genome editing by CRISPR/Cas9 [4][5][6][7]. Mutant screening by PCR is efficient for multiple samples. Escherichia coli colony-PCR is also an efficient technique for screening positive colonies [8][9][10], 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][14][15]. 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 [16,17]. 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 [16,17]. 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.

Experimental Design
A manual centrifuge was developed to spin solutions in 96-well PCR plates with a base to fix two 96-well PCR plates that can be centrifuged simultaneously. The improved salad spinner can be used as a simple centrifuge machine with a speed of ~700 rpm and functions efficiently with 96-well PCR plates for a short spin. A conventional electric centrifuge was used to compare the characteristics of the manual centrifuge.

Materials
Polystyrene foam (200 × 200 mm, thickness 25 mm) was procured from the lid of a reagent delivery box and used as the base for fixing two 96-well PCR plates. A salad spinner (Pearl Metal Co., Ltd., Sanjo, Japan; Cat. No.; Petit chef Jr C-750, http://www.kitchen-tool.com/goods_C-750.html, https://amzn.to/2AkQcN0) was used as the body of the manual centrifuge to spin solutions down in 96-well PCR plates. A semi-skirted 0.2-mL 96-well PCR plate (Nippon Genetics, Tokyo, Japan; Cat. no.; 4Ti-0760) was used to spin solutions down with the manual and electric centrifuges. EZ-Vision One DNA 6× loading buffer (VWR Life Science, Radnor, PA, USA; Cat. No.; N472-KIT) was used to stain DNA after agarose gel electrophoresis [19].

Assembly of the Manual Centrifuge
The processed polystyrene foam base was assembled with the body and centrifuge basket ( Figure 2A). Figure 2B-E show the parts and assembled centrifuge used to spin solutions down in the 96-well PCR plates.

Equipment
The MPS 1000 Mini Plate Spinner Centrifuge (Labnet International, Inc., Cary, NC, USA; https://www.labnetinternational.com/products/laboratory-centrifuges/mini-centrifuges/mps-1000mini-pcr-plate-spinner) was used as the control electric centrifuge to spin solutions down in 96-well PCR plates in comparison with the manual centrifuge developed in this study.

Procedure
Centrifuging Solutions in 96-Well PCR Plates before DNA Agarose Gel Electrophoresis (Time for Completion: ~5 min) 1. Add 2 µL of EZ-Vision One to all PCR samples (10 µL) in 96-well PCR plates. 2. Set two 96-well PCR plates on the base of the manual centrifuge ( Figure 2D).

CRITICAL STEP:
When centrifuging one 96-well PCR plate, another should be placed on the opposite side for balance. 3. Rotate the handle of the salad spinner for 3 s. 4. Check that the PCR mixtures have settled to the bottom of the wells in the 96-well PCR plate and load samples for agarose gel electrophoresis [19]. OPTIONAL STEP: The centrifuge can also be used to spin the PCR reaction mixtures down in 96-well PCR plates before PCR.

Expected Results
PCR solutions in 96-well PCR plates were sufficiently centrifuged using the manual centrifuge for 3 s ( Figure 3A). DNA fragments from these centrifuged PCR mixtures could be clearly visualized by DNA agarose gel electrophoresis (Figure 4) [19]. In contrast, using the conventional electric centrifuge resulted in poor centrifugation after 3 and 10 s (Figure 3B,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 3D). 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) [16,17]. 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.  [20], and visualized by a combination of a black light and a longpass emission-filter (SC-46, Fujifilm, Tokyo, Japan) [19]. This study [16] [18] Table 2 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.