User:Norman Wang/Blue Light Transilluminator

• Build a fluorescence Transilluminator suitable for visualizing SYBR Safe DNA dye. For gel-band-cutting without DNA damage nor need for UV protective gear.
• For visualization/photo documenting with no downstream use of the DNA, UV excitation of SYBR Safe dye is preferred. Because the UV excitation (a secondary peak) and emission peaks are further apart and facilitates better wavelength separation.

Methods

Dye of interest: SYBR Safe (possibly other related generic brand SYBR dyes)

SYBR Safe Excitation (dotted) Emission (solid)

Attempt #1

• Excitation Source
  • 470nm LED Lights
  • Shortwave UV
• Visualization Post-filter
  • Amber Filter for Camera
  • Orange SYBR Safe glasses from Invitrogen

Attempt #2

• Wratten 47B or Wratten 47 Filter for filtering blue LED light

Results

Attempt #1

• Illumination with Blue light without excitation pre-filter seem to have green light leaking through, hence the overall green background glow (SYBR Safe has peak excitation of 502nm and 530nm emission which are quite close to each other). Whereas under UV excitation there is little background green glow (UV <300nm light is completely blocked by orange filter)

• 

  excitation using 470nm Blue LED (no deep blue pre-excitation filter), visualized behind amber postfilter

• 

  excitation using Shortwave UV, visualized behind amber postfilter

Attempt #2

• Use Kodak Wratten 47 or 47B filter to pre-filter excitation blue LED light source and visualize under the same Orange/Amber emission post filter.

• 

  Filtered using Kodak gelatin filters 47 vs 47B (un-annotated image)

• 

  Unfiltered 470nm LED lights, background green is washing out the fluorescence from SYBR Green dye.

Absorbence Curve of Wratten 47 vs 47B filters

Discussion

Wratten 47 Filter is Better

Wratten 47 vs 47B side by side comparison shows that although 47B is darker blue, it leaks green light where 47 blocks it. I suggest we purchase Kodak Wratten 47 (tiffen glass) to build the proper translluminator box.

Conclusion

• SYBR Safe dye is not as good at EtBr in terms of brightness (given my current setup), although it offers several advantages.
  1. Safer
  2. Cheaper disposal
  3. Visualize using blue light instead of UV
• Future improvements
  1. add/concentrate LEDs for brighter light box
  2. increase SYBR Safe concentration (eGel contains minimal amount of SYBR Safe, Invitrogen cutting cost?)
  3. characterize amber filter and select an appropriate off-the-self amber light filter. Contact me if you have any suggestions.

Suggested Usage

Blue Light + SYBR Safe

• for Clone well technique to extract pure DNA straight from well (where undamaged DNA is essential)
• for quick gel runs to get yes/no product & band size verification
• for cheap eGEL boxes
  • no loading dye is needed
  • less agarose used (~20mL gels)
  • less TAE buffer used (~5mL Acetate + ~5mL Tris)
  • possible to precast as many gels as the eGel holders (instead of waiting 40 min each time for gel to solidify)

GFP Excitation

• less damaging to live cultures expressing GFP

Used Ethidium Bromide Where It's Best

UV + EtBr

• EtBr is by far cheap in cost, but disposal and environmentally expensive
• for taking publication quality photos

Blue Light Transilluminator Under $100

My 5min hack a blue-light box setup

• $80 60 LED Array (15LEDx4)
• $10 Used Kodak Wratten 47 Gelatin Filter on ebay, new $20-60, Lee filters can be purchased for much less although I have not measured its spectral characteristics.

‡ Smaller scale transilluminator with less LEDs can be built for much less.
† Colored acrylic may be used to reduce cost
∞ Tiffen glass light filters can be used where durability is an issue and cost is not.

Any comments, suggestions or improvements, please contact Norman Wang