Dear All, I'd like to thank everyone who responded to my query. I haven't had the chance to try all of the suggestions, but I have had some success (all posted below). I've tried the following without success (in addition to the list on the original post): - DMSO - BSA - Gene Releaser - DNA Ligase (not conclusive yet) (in combination or isolated, different volumes, etc.) The only thing that seemed to work for some samples was incubation in Proteinase K for 2 hours at 60ºC. There seems to be also an additional quality problem that was not evident in the beginning. After denaturing the re-digested samples at 95ºC for 5 minutes I ran them on an agarose gel again and they looked smeary (degraded). It became obvious the double stranded DNA had nicks. I incubated a bunch of samples in different conditions to test for endonucleases and that did not seem to be the case. The best explanation I have so far would be degradation by some physical agent. These DNA samples were shipped by FedEx and might have been X-rayed. Has anyone had any problems with X-rayed DNA samples before? Is there a better and safer way to ship DNA samples? Thanks, Hugo This was the original post: I am having trouble getting PCR amplifications from some of my samples. I have not access to more tissue sample, so I have tried re-cleaning the genomic DNA in solution using different methods with *very* little success, usually losing much of the DNA in the process. I've tried: - Phenol-chlorophorm-isoamyl + chlorophorm-isoamyl + ethanol precipitation; - Heating them in NaOH+SDS for 20 minutes (following Shi et al. 2004) followed by Phenol-chlorophorm-isoamyl + ethanol precipitation; - Promega Wizard Genomic DNA Purification kit; - Qiagen DNeasy Tissue kit; - Qiagen DNeasy Plant kit. I have plenty, long non-degraded DNA in solution (checked on Nanodrop and on agarose gel). I've also tried diluting the samples with no success. When co-amplifying these samples with ones that work, I still get amplifications (that sequence well) which means whatever it is, it's not something in solution but most likely bound/cross-linked to the DNA. I must add that these samples were originally extracted from fish muscle tissue preserved in ethanol or frozen, using an ammonium acetate protocol and isopropanol precipitation. I'd be grateful if you could suggest some alternative method that has worked for you, including modifications of the above (as they are typically meant for extraction from tissue and not from aqueous solution). A compilation of mostly unique answers: I'm not sure what taxa you're looking at but have you tried CTAB in the initial step. We use it with molluscan tissues and it seems to take out the PCR inhibitors. >>>>>> probably you have some proteins in your DNA extraction that inhibit PCR amplification. You could try to add some small amount of Proteinase K to your solution. This breaks up these proteins. We use Qiagen Proteinase K of 10-20µl (at the moment i have my Labbook not here, so i can´t tell you the concentration, but i could look up). Then use 50-60°C for about 30min till 3h and then retry the amplification. An other point is to add more Taq-Polymerase or use a better Polymerase, like Phusion (sometimes you have to raise the concentration of MgCl2 in your PCR cocktail then). Normally this will help. Good luck, >>>>>> Is there a difference in molecular weight/quality of the DNA that works versus the DNA that does not amplify? Depending on the size you are attempting to amplify, something else you could try, unrelated to DNA purification, is cutting your DNA with a restriction enzyme. I've had limited success doing that when I have problems amplifying extremely high MW DNA samples. You might also try an extra long denature on your PCR cycles to see if that works. >>>>>> you might try treating the extracted DNA (from any of the methods listed) with (v/v) 1:1, 1:2 or 1:3 10% Chelex resin (BioRad). Basically, for 1:2, you mix 1 part DNA with 2 parts 10% chelex, heat to 100 C for 10 minutes, spin down and use the supernatant in your PCR reaction. You can scale this up to 96-well (or greater) by filtering the DNA+Chelex through a millipore plate to remove the chelex from solution (it can inhibit PCR). this works well for us with problematic templates (DNA extracted from blood, tissues, feathers, etc.). In fact, we treat all of our samples this way, since it is inexpensive. you might also give the DNA Clean and Concentrator (Zymo) kits a shot, but that is probably not going to have a terribly different effect from running another Ethanol precip., although you may not lose quite as much DNA. >>>>>> How much have you tinkered with the PCR reaction? You've probably tried this, but are you using Bovine Serum Albumin (BSA) in your PCR reactions? It non-specifically binds to just about everything except DNA and if you're not using it, you should try it (final concentration 1ug/ul). You could also try adding some DMSO, which helps separate the DNA strands and holds them apart (in addition to the BSA). I've found using these, particularly the BSA, is critical when working with samples where various contaminants are bound to the DNA. >>>>>> I've had some luck cleaning up DNA extractions using the Qiagen PCR purification kit. I know it sounds strange to run genomic DNA through a PCR kit, but I gave it a try thinking that it was designed to deal with an aqueous solution (plus, I was desperate!). I've tried this method to clean up genomic DNA extracted from rotten tissue. I have been able to amp from the cleaned genomic DNA about 70-80% of the time. Checking the before and after genomic DNA on the Nanodrop also indicates that the after sample has less RNA and other impurities than the before sample. Hope it works for you! >>>>>> To test whether there is fact a PCR inhibitor in your samples (that would definitely need to be cleaned out somehow), have you tried the following test? Set up a PCR reaction for three samples. In the first tube, use a sample that is known to work (i.e., positive control) . In the second sample, combine the positive control template and your problematic sample. In the third tube, use just your problem sample as the template. If your problem sample contains a PCR inhibitor, then it should "shut down" amplification of the positive control in the second tube (and, of course, the third tube with just the problem sample should not work either). For some problem samples in my lab, we have done a second salt/ethanol precipitation using 5 M lithium chloride as the salt and that has helped get rid of PCR inhibitors. You will lose DNA yield though. >>>>>> I am having trouble getting PCR amplifications from some of my samples. I have not access to more tissue sample, so I have tried re-cleaning the genomic DNA in solution using different methods with *very* little success, Looks to me as though you have tried everything that can be expected to work, in which case priming-site divegence seems a possibility. But you do not say whether you have tested these DNAs with other primers, nor what primers you have used. I would test the DNAs with a selection of closely spaced SSU, LSU or mtDNA SSU primers and if these also fail the DNASs must be nicked. If they succeed then your other (?specific gene) primers do not match these samples. The only other possibility I can think of is that these DNAs might need very extended protease digestion before purification. >>>>>> as you get amplification if you add good DNA to the samples, it seems that the problem is not due to inhibitors, but to degradation of DNA. This does not need to be visible on gels, but you can have plenty of single strand breaks in the DNA backbone. The double strands will still hold together which is why you see apparently long DNA on gels. You did not say anything about the source of your material, but I assume it might include old DNA (from herbaria or museum specimens). I attach two files that may help. One is a file you can open with any web browser, the other is a paper from the same author. We have tried this, but with limited success, but maybe you are more lucky. Another idea is that you may try to add ligase in an appropriate buffer to your DNA and incubate it. Maybe this will repair the strands (if that is the problem). Good luck! >>>>>> One things you might try is Gene Releaser: http://www.bioventures.com/_pdf/GeneReleaserTBRevA1.pdf?PHPSESSID=3Dqxozgisqv >>>>>> Don't think it is the way you initially store tissue or extract DNA as that should be fine. 1) I would try PCR cleaning/concentration kit. We use 'Genetix' DNA Clean and Concentrator. It is a membrane kit, indeed starting with aquaous DNA (for example after PCR). 2)dna concentration could be too high. Have you tried PCR of same stuff but then diluted 10 or 100 x in H2O? 3) Have you Rnased the DNA solution? Could RNA interfere? >>>>>> You may like to try 'Millipore - Microcon DNA concentration tubes', I used them a long time ago with success, and a colleague of mine routinely uses them. They not only concentrate DNA but are supposed to remove various things that can act as PCR inhibiters. The capture rate is quite good, and the filters can be inverted and back-spun with the addition of buffer if the membrane has spun dry. There is quite a lot of review info on the web. >>>>>> One thing you could perhaps try is Chelex extraction. Boiling the samples may break down some of the inhibitors that are causing problems, and the beads absorb any ions that may be causing amplification problems. If that doesn't work perhaps try whole genome amplification. The REPLI-g kit from Qiagen, for example, is very easy to use and surprisingly cheap, and will give you the concentration you need to run a normal PCR. >>>>>> Could there be a PCR inhibitor in your sample. Try adding BSA or DMSO, or both as part of your PCR. Many of my samples don't work because of this. Also try a 1:10, 1:50, 1:100 dilution of your sample - this also helped me. Try this with and with and with out the BSA/DMso. >>>>>> Just a little suggestion from my side: Have you looked into magnetic neads purification? When it comes to purity issues these are used with great success. I use magnetic beads on viral RNA from faecal samples, probably containing really bad PCR inhibitors. I've never used it on genomic DNA, though, so please don't expect that it works right away. A quick google pointed me to http://www.biocompare.com/Articles/ProductComparison/157/Magnetic-Beads-for-Genomic-DNA-Purification.html, but you can try your own google search. I'd use 'magnetic bead genomic dna purification' as search term. Hope to have shown you another option, >>>>>> Hugo F. Gante Hugo.Gante@asu.edu School of Life Sciences Arizona State University Tempe, AZ85287-4601 and Museu Nacional de História Natural Rua da Escola Politécnica, 58 Lisboa, Portugal hgante@asu.edu