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Old 11-21-2018, 04:14 PM   #1
Colaptes
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Default UV Transilluminator?

Hi all,

I'm preparing some GBS libraries for Illumina sequencing and have a quick question about gel extraction:

In my old lab we used a blue box transilluminator for cutting bands out of gels for size selection, but I've moved to a lab that only has a UV transilluminator. Will it damage my sequencing library if I expose the gel to UV light while cutting the band? I am concerned about degrading the library or introducing mutations that could get called as SNPs in downstream analyses.

Thank you!
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Old 11-21-2018, 11:08 PM   #2
nucacidhunter
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It will damage but I am not sure to what extent. You can work around it by staining and marking target sizes on the marker lane cut from the gel and using it as a guide for cutting target region from samples.

UV induced mutations should not be an issue as it should happen in all reads of the same tag from the same sample to result in false call. The coverage will take care of it.
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Old 11-22-2018, 11:11 AM   #3
ATϟGC
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Since a lot of dsDNA fluorescent dyes have an excitation maximum of about 490 nm (blue light) and and emission at about 520 nm (green light) You can make your own blue light epi-illuminator to excise the bands for about $30 Canadian. You need the following 3 things:

1=Blue light source:
I have done this by buying a cheap LED flashlight with a single LED bulb at one of the "dollar" stores for $2 or $3 and then I took out the LED and resistor and replaced it with a Blue light LED (~490 nm wavelength $2 or less each) and the appropriate resistor. Alternatively, It might be easier to find a blue flashlight or headlamp that emits blue light on amazon or in a deparment store. Anywhere in the 460-500 nm range should be fine to excite the dye. The Rayovac sportsman headlamp or flashlight has blue LED that might work, although I have not tried them.

2=Orange Filter:
You need to filter the blue light so that you can see the green fluoresence of the DNA on your gel. I use inexpensive orange safety glasses. Here are two I have used with success:

https://www.amazon.ca/Uvex-S1933X-Ey.../dp/B000USRG90

https://www.amazon.ca/Uvex-S0360X-Ul.../dp/B003OBZ64M

3=Fluorescent dye with excitation of ~490 nm and emission at ~520 nm:
Just about any of the blue light transilluminator-friendly dyes should work (i.e. Gelgreen, Sybrsafe, Sybrgreen).

I have even made a trans-illuminator cheaply with a 20 cm square sheet of blue plexiglass and orange plexiglass or the orange glasses mentioned above. You just put a diffuse white light source below the blue plexiglass, put your gel ontop of the blue plexi and then filter out the green with your glasses or a sheet of orange plexiglass. The glasses are better as they are hands free.
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Old 11-25-2018, 09:29 AM   #4
Colaptes
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Thank you for the tips! I will try the workaround marking the ladder cut from the gel, and look into making a blue light transilluminator for the future.
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Old 11-26-2018, 07:40 AM   #5
pmiguel
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Yes, please everyone switch to blue light illumination for preparative gels! We did so > 20 years ago. I'm just realizing that many labs (and new PI!) are unaware of the problems UV light damage to DNA can cause for downstream processes.

For us about 1 bizarre customer sample issue per year gets traced back using a UV transluminator for prep!

If you are a new PI don't buy a UV box. Just buy a "dark reader" blue light transluminator. Or just jury-rig one up using AT-GC's recomendations. (If it works for you -- you may not have any Canadian dollars...)
--
Phillip
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