Molecular Cloning: UV Damage - (Feb/27/2006 )

so I've been cloning for about 3 years now with average success (usually takes 3-4 tries for anything - and generally only about 30-40 positive clones).. I recently ran into a cloning impasse where absolutely nothing would work. as usual, I searched in and out through the mol bio forums for possible solutions... I stumbled upon one key point I haven't paid much attention to in the past.. UV damage to DNA by the transilluminator. We'd recently acquired a new one and, since then, I haven't turned down the UV for preparatory gels. To test whether UV damage may be my problem, I replicated a cloning protocol and extracted the fragments without ever exposing them to UV. I did this by simply running a small amount in a neighboring lane, slicing the entire lane out then cutting around the reference band... I then lined it back up with the elution lane and cut appropriately. Processed both vector and insert this way. <I should also note that I typically cut slices out very quickly, so it's not that I've left my gels on the UV for hours!>

End result... hundreds of positive clones... the same strategy done with my normal (over UV) extraction yielded 0.

I suppose most people get around this by excising over a glass dish .... but when dealing with difficult cloning strategies where every small decrease in efficiency matters... maybe best to avoid UV altogether.

anyhow... figured I'd give something back for all the times this forum has gotten me out of troubles and post a heads up that some might find useful.

best.
ralfa

It's not so much the UV light, but the intercalated EtBr in conjunction with UV light that damages the DNA.

The simple way around that is to not add EtBr to your gel and the buffer in which you run your preparatory sample, just incubate the gel in diluted EtBr for a very short time after it is run, so only the outer layer getss stained. This way you'll see the band under UV light but most of your DNA will not be stained with EtBr and therefore not get damaged that much.

LeserattePD

We add guanosine to gels from which we recover DNA for cloning. It acts as a UV protectant -- from old BioTechniques article.

The DarkReader (blue light, yellow filter) eliminates this problem, while being compatible with EtBr staining.

we use a methylene-blue staining protocol for preparative gels:

1) you don't have problems with EtBr

2) you don't need UV-light but normal white light to see the dna fragment

the only thing is....its not that sensitive. You need a good amount of dna to refind it under white light.