I received a number of answers regarding the UV sterilisation of PCR and extraction reagents. Many people advocated the use of filter tips and small aliquots of reagents but the actual use of UV received a variety of responses; I used UV sterilization to avoid a persistent contamination in our lab.I sterilise pipettes, tips, tubes and water under UV for 20 min. Then Iperform my PCR adding everything (primers, dNTPs, water, Mg) but the Taqand DNA and then I sterilise the master mix for 10 min. Then add Taq andDNA. With this protocol I manage to avoid the contamination. The amountof PCR product was a bit lower than without the UV treatment but it wasgood enough.I never tried to sterilise the Taq but I'm not sure is a good idea. WithdNTPs there is not any problem and with primers there is not problems ifthey don't have several T together (they could form T dimers, that caninhibit the PCR).I send you a paper with some information about UV sterilization (Padua RA, Parrado A, Larghero J, Chomienne C (1999) UV and clean air result in contamination-free PCR. Leukemia 13, 1898-1899.) Good luck Javier Montero-Pau Javier Montero PauLaboratorio de Ecología EvolutivaInstitut Cavanilles de Biodiversitat i Biologia EvolutivaUniversitat de ValènciaAO. 22085Valencia I wouldn't expose dNTPs to UV light. UV light excites DNA. In yourcells, UV light causes mutations and cancer. We use UV light to"sterilize" things used in pcr because it damages DNA. ad dNTPs arethe components of DNA, they are also likely to be damaged by UV light. I don't know about the enzymesbestdiana Hi Claire, I would suggest several things: Using a negative control (no DNA added) for each PCR will alert you to possible cross contamination. If available, using filter tips will help to minimize cross-contamination. Cleaning your work space and pipettes daily, and making small aliquots (single or a few uses) of your reagents (dNTPs, primers, water, etc) will also help to minimize risk of contamination. You can order small bottles of double distilled water (if this is not available to you at your institution) for resuspending primers and for making your master mix (if you order PCR kits, they often come with clean water). You mentioned sterilizing your Taq and dNTPs and this strikes me as not necessary at all. These two reagents should not be left at room temperature (especially Taq!) for any length of time (I always keep mine in the -20C freezer at all times in a enzyme cooler) and they should arrive free of any DNA (especially free of the DNA you are working with and are concerned with). So exposing them to UV light seems unnecessary. Good luck! Nikki Freed nicole.freed@env.ethz.ch Best and safest thing to do is to use separate sets of tips, pipettes,reagents, etc. in different rooms for all pre-PCR reactions. I've heard(and read) it was not good to UV-sterilise PCR tubes, as plastic suffersfrom it, and I've never UV-sterilised reagents. Title: UV light irradiation of plastic reaction tubes inhibits PCRAuthor(s): Burgess, LC; Hall, JOSource: BIOTECHNIQUES Volume: 27 Issue: 2 Pages: 252-+ Published: AUG 1999 Violeta Muñoz. Hi Claire,UV sterilizing enzymes will kill them so that is not a good idea.You should try to find a hood somewhere else (i.e. in a lab that doesnot work on this species or anything related). It is also best if you dothe work in a building or part of the building that does not share thesame ventilation system. Basically, museum specimens are consideredabout the same as "ancient DNA" so you really need to use the same sortsof precautions. This includes using extraction and PCR blanks to testyour reagents, ordering all reagents, gloves etc. separately (and do nottake them into the lab where the modern specimens are), and avoidinggoing into the modern lab before you go to the other lab to work on themuseum specimens. It might also be best to do the museum work first.Finally, depending on the age of the museum specimens, you might need todesign your primers such that they amplify short (100-200 bp) fragments. Good luck!Anne UV treatment of liquid reagents in a hood (with a UV bulb) is not an idealsterilization method - as i understand it, certain kinds of plastic blockUV rays, and UV of this strength can only penetrate in liquid a very shortdistance - maybe a centimetre at best (imagine how colours change underocean waters - and that's full-strength solar power!). You're basicallyjust wasting energy and time. And blasting chemicals with bond-breakingrays makes me nervous. For reagent sterilization, 0.2 micron filter units are best - you can uselarge filter units for larger volumes (250ml+) and syringe filters forsmaller volumes. However, I've worked with herbarium specimens also, and with good steriletechnique, had no cross-contam problems. Remember that most molecularreagents come pre-sterilized, so re-treating them has more potential tointroduce contaminants. Take good care of your reagents by pre-aliquotingthem into single-use (like one days' worth) volumes, treat your samplescarefully, and you'll be fine. Good luck!~a -- Amy Smithmasmith@nature.berkeley.eduEnvironmental Science, Policy and Management137 Mulford Hall - #3114UCBerkeleyBerkeley, CA 94720-3114510-642-9013(tel) I don't think anybody does UV sterilization of enzymes, although I am not sure the dosage you want to deliver would be sufficient to crosslink and reduce the efficiency. I would still like to know what other people think.May be you should look up some ancient and forensic DNA isolation procedures and you may find some clues regarding cross-contamination issues.Ancient DNA: http://search.vadlo.com/b/q?sn=3D158621799&a...ation&rel=3D0Forensic DNA: http://search.vadlo.com/b/q?sn=3D158621799&a...c+DNA&rel=3D0Museum specimen DNA: http://search.vadlo.com/b/q?sn=3D158621799&a...ation&rel=3D0 UV can be used to sterilize liquids, depends on wavelength, power of UV-source, turbidity of liquid, thickness of liquid layer, etc. how long it should be irradiated.For dNTPs I would not use it, they are much too susceptible. Proteins may be denaturated (?), depends perhaps on their stability, but proteinase K is very stable, I guess it can stand it. Most buffers anyway. But PCR reagents are sterile (or were, before use), so there is no need to sterilize them. If they are contaminated with DNA, MOs, etc I would replace them. From: claireraisin@hotmail.com To: evoldir@evol.biology.mcmaster.ca Subject: Other: Use of UV for sterilising reagents? Date: Thu, 22 May 2008 10:25:14 +0000 I am based in a molecular ecology laboratory and am planning to extract DNA from and genotype modern and museum specimens of the same species. So, obviously there is potential for cross-contamination here. I work under a UV hood and so all my equipment is sterilised under a UV crosslinker before use. I also UV sterilise my extraction buffer and ddH2O, I was wondering if enzymes (such as proteinase K and Taq) and other PCR reagents (i.e. dNTPs) can also be treated in this way or would their efficacy be adversely affected? Some people have suggested that as proteinase K is so stable it should be able to withstand UV but that dNTPs could not, others disagree! Any suggestions? Many thanks, Claire claireraisin@hotmail.com