UV light, dangerous to purify PCR fragments? - (Feb/04/2007 )
I have different suggestions from my previous and current lab concerning using UV light to cut out the desired band from agarose gel which will then be used in subsequent cloning reactions.
I would like to hear your comments and reasoning about this topic.
UV is bad for your DNA. Both in your gel and in the cells of your skin. And as I understand it, UV is also bad for your eyes. It makes the cornea cloudy giving rise to premature cataracts.
If you can, get a blacklighter. There is a thread on making your own blacklighter cheaply somewhere on this forum.
A blacklighter is simply a transluminator that shine blue light (of a certain frequency) and a filter over the top that blocks out blue light of that same frequency. Ethidum bromide absorbs blue light and floresence orange. (usually this is too week to see as it is swamp by the blue light sourse.) But with a blue light filter, the orange glow can be seen. The same thing happens with UV, except that our eye can not see UV.
But if have to use UV like most of us, minimise exposure. Think in time of seconds. So work fast and work quickly. (wearing face mask, gloves, labcoat and all) You don't want sunburn, or skin cancer 25yrs from now.
And there are two kinds on UV transluminators, the short wavelenght and long wavelenght (and also the tuneable variaty). You want the long wave lenght, less energy and less damaging to DNA. (TT dimer formation) but harder to see the DNA bands (somewhat). Make sure you have the right one.
Nevertheless, the my supervisor say DNA that has been exposed to UV transforms 10 times less efficiently. However I tend to use buckets of DNA in my ligation and thus have not noticed any difference. The story is different however with large plasmids, Bac and PAC (>20kb) Apparently the effect is real. So my other labmates conduct surgary on the gels. After running the gel, they cut out a frame, leaving a little of the DNA band as a sacrifice on the frame. The frame is UV, and the location of the desired DNA band is marked on the frame. Then the frame is placed back and using the marking on the frame, cut out the band of interest.
And thus the DNA bands is cut without exposure to UV.
UV-generated damage to DNA stained with ethidium bromide reduces the transformation efficiency. One minute of exposure to 312 nm UV can reduce the transformation efficiency by > 90%. (BioTechniques 1999, 11:747-748 and BioTechniques 1996, 21:898-903) UV-damaged DNA products include cyclobutane pyrimidine dimer (T-T), 5-thyminyl-5,6-dihydrothymine, 4,6-diamino-5-formamidopyrimidine, 5-hydroxy-5,6-dihydrothymine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 6-hydroxy-5,6-dihydrocytosine and 8,8-adenine dehydrodimers (from poly(dA). Guanosine is reported to protect DNA from damage by UV transillumination. 1 mM guanosine added to the gel and the running buffer “increased the yield of clones by a factor of about 400, compared with conventionally prepared, unprotected DNA” exposed to 312 nm UV. (D. Grungermann and E. Schomig, 1996. Protection of DNA during preparative agarose gel electrophoresis against damage induced by ultraviolet light. BioTechniques 21(5):898-903.)