High-sensitive and low-cost DNA agarose gel electrophoresis detection system

icon Developement of highly sensitive and low-cost DNA agarose gel electrophoresis detection system

 We developed two DNA agarose gel imaging systems (ref. 1). An ultra-low-cost system utilized black light as an excitation light source with a commonly used photography filter as an emission filter. A different low-cost system was able to detect DNA bands at the same sensitivity as a UV-ethidium bromide system. The system can be used with a comcombination of cyan-LED light, a shortpass excitation filter, and a longpass emission filter. Digital imaging parts for image-recording can be shared between two excitation light systems.
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A. Digital imaging parts (for both black light and cyan-LED systems)
1. Digital Camera
Canon PowerShot G12
※A smartphone camera may also be used.
2. Copy stand
Copy stand (LPL, CS-A4)
3. Blackout curtain (or cardboard box)
Blackout curtain (100% polyester, ~ 315 g/m2, 3 m)
※A cardboard box may also be used as an alternative for the blackout curtain (see photograph).

Photographic conditions: ISO 400, F4.5, 1-2 sec., no frash
 
 [Features of the digital imaging system]
・This imaging system can be used for agarose gel extraction with specific color spectacles as agarose gels can be placed in the open space (see photograph).
※ Yellow spectacles (TRUSCO, TSG-814Y) may be used for EZ-VISION, Safelook Load-White.
※ Orange spectacles (UVP, UVC-310) may be used for Midori Green Direct, Safelook Load-Green, and SYBR safe.
 
   

Figure 　DNA agarose gel detection system.
 
 
Figure Schematic diagram of the DNA agarose gel detection system using a vertical illumination technique.
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B. Excitation light system
B1. Ultra-low-cost DNA agarose gel electrophoresis detection system using black light.
※This black-light excitation system can be used for DNA agarose gels stained with EZ-VISION (VWR) and Safelook Load-White (Fuji film Wako Pure Chemical).
1. Light source
Black light blue lamp 27W (360 nm, FPL27BLB, Sankyo Denki)
Fluorecent lamp stand (LK-H451W, Twinbird)
2. Emission filter
SC-46 (75 × 75 mm, Fuji film)
3. Yellow spectacles (for gel-extraction)
Yellow spectacles (TSG-814Y, TRUSCO)
[Features of the excitation system using black light]
・The black light system, which can excite EZ-VISION, is an ultra-low-cost vertical illumination system, compared with a UV-transilluminator (> $1,000).
　※This system (black light and emission filter) can be purchased for $100 or less.
・DNA agarose gels can be photographed using a standard digital camera (see photograph below).
・EZ-VISION (VWR) is a non-toxic reagent, however, EZ-VISION is less sensitive than ethidium bromide.

Figure DNA agarose gel electrophoresis visualized by EZ-VISION and black light
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B2. Highly sensitive and low-cost DNA agarose gel electrophoresis detection system using cyan-LED light.
※This cyan-LED excitation system can be used for DNA agarose gels stained with Midori Green Direct (Nippon Genetics), Safelook Load-Green (Fuji film Wako Pure Chemical), or SYBR safe (Thermo Scientific).
1. Light source
Cyan-LED (490-495 nm, hlk-12led-490-495nm, Holkin)
2. Excitation filter
SV0510 (50 × 50 mm, Asahi Spectra)
3. Emission filter
SC-54 (75 × 75 mm, Fuji film)
4. Orange spectacles (for gel-extraction)
Orange spectacles (UVC-310, UVP)
 
[Features of excitation system using cyan-LED light]
・Usage of Midori Green Direct, Safelook Load-Green, and SYBR safe indicated sensitivities equal to ethidium bromide staining (ref. 1 Figure 1). The sensitivities of these staining methods were ten-fold higher compared to the EZ-VISION method. However, Midori Green Direct is more expensive than EZ-VISION. We use either EZ-VISION or Midori Green Direct, depending on the experimental setup.
・Many commercially available LED systems use blue-LED (470 nm). However, cyan-LED (490 nm) can efficiently excite Midori Green Direct, Safelook Load-Green, and SYBR safe, even more than blue-LED (ref. 1).
・The components of this system (cyan-LED, Ex-filter, and Em-filter) can be purchased for $280.
・This imaging system can be used for agarose gel extraction with orange spectacles as agarose gels can be placed in the open space (see photograph).
 
Figure DNA agarose gel detection system by cyan-LED light (photograph)
[Blue-LED light as an alternative to cyan-LED light] 
・This system can also be used by excitation with blue-LED light alone, however, the wavelength of blue-LED is not optimal. As a simple method, the blue-LED light system can be used for DNA agarose gels stained with Midori Green Direct ($100 - $200; ref. 1). However, the blue-LED light is less effective compared to the cyan-LED + Ex-filter system (ref. 1).
・The costs of blue-LED lamp are being gradually reduced. Sometimes, we can purchase blue-LED lamps which can be used for this system for several dollars online at Banggood.com.
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 [Light source and filter]
　1) Combination of light sources and DNA-staining reagents used in our laboratory
   Black Light & EZ-VISION;  SC-46 (Em-filter)
   490 nm LED light (+Excitation shortpass filter) & Midori Green Direct;  SC-54 (Em-filter)
    ※Sensitivity: Midori Green Direct (Nippon Genetics) ＞ EZ-VISION (VWR)  
    ※Running cost: Midori Green Direct (Nippon Genetics) ＞ EZ-VISION (VWR)  

　2) Combination of other light sources and DNA-staining reagents
   Black Light & Safelook Load-White;  SC-46 (Em-filter)
   490 nm LED Light (+ Excitation shortpass filter) & Novel Juice;
　　　※Novel Juice is less sensitive than Midori Green Direct, and Safelook Load-Green.		  SC-54 (Em-filter)
   490 nm LED Light (+ Excitation shortpass filter) & Safelook Load-Green;  SC-54 (Em-filter)
   490 nm LED Light (+ Excitation shortpass filter) & SYBR safe;  SC-54 (Em-filter)
　3) Information on DNA-staining reagents, light sources, and optical filters
DNA staining Ex Em Light source Emission filter
EZ-vision 364 nm 454 nm Black Light (360nm) SC-46
Safelook Load-White 370 nm 470 nm Black Light (360nm)  SC-46
Midori Green Direct 490 nm 530 nm CyanLED (490 nm) + >510 nm cut SC-54
Novel Juice 495 nm 537 nm CyanLED (490 nm) + >510 nm cut SC-54
Safelook Load-Green 490 nm 525 nm CyanLED (490 nm) + >510 nm cut SC-54
SYBR safe 502 nm 530 nm CyanLED (490 nm) + >510 nm cut SC-54
  ※ Running costs of DNA-staining reagents are described in ref. 1 (Table 2).
  4) Costs of excitation light systems
Commercially available UV-transilluminator, 　>$1,000
Commercially available blue-LED light system,   $600 - $1,000
Blacklight system,  <$100
Cyan-LED system with excitation filter,  　 $280
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[References]
　ref. 1 Ken Motohashi
"Development of highly sensitive and low-cost DNA agarose gel electrophoresis detection systems, and evaluation of non-mutagenic and loading dye-type DNA-staining reagents"
PLOS ONE 14, e0222209 (2019) 　
 
         
 
 
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