Yes. At least I had such results. I work on moths. All the specimens are killed using ethyl acetate. Even though high DNA content but did not show bands on electrophoresis was spectrophotometrically observed, no results for PCR were obtained

I am also working on moths and confronting same problem in some specimens but some time results are good too. so in your case PCR I think you should adopt more primer, I took few couple of primers and found good results so its not sole killing agent responsible for this problem some time primer, age of specimens and postmortem storage of specimens are also responsible. you can also visit to this publication

Influence of killing method on Lepidoptera DNA barcode recovery 2014.

Thank you both for your answers. Aditi Shere-Kharwar I was also wondering about storage. If your specimens were stored at room temperature and analysed tissues were not fixed in ethanol, that could be the reason for poor PCR success.

@Matic first of all pardon my poor language drafting in last message I am not sure what happened. It's commendable that you even understood what I wrote....

Coming to your point. Yes my samples were not stored in ethanol at that time. Although this was my very first experience. Since then, while collecting I sedate the moths and procure their leg(s) and then preserve the specimens by oven drying. The tissue procured is stored in absolute alcohol and is kept in deep freeze. Now even after a decade I am getting positive results from them.

@Santosh did you get good results for small moths? My observation is small moths die faster so their exposure is reduced. It's an established fact that ethyl acetate causes DNA damage. Thus, lesser the exposure better chances of getting good results.

Further, I got results for PCR if the DNA quality and quantity was good. Whenever PCR failed normal troubleshooting was able to get results. Well age of tissue sample has not hindered any results when stored in deep freeze under absolute alcohol.

@ Aditi Shere-Kharwar I am working on Lithosiini moths & majority of moths in this tribe are smaller. better you take some more primes.

Yeah sure

Matic Gabor,

Given the international alcohol carry and export rules of vinegar is recommended as a standard fluid for transportation of soft bodied taxa. I have never used it. However, Vinegar contains acetic acid, which during DNA extraction neutralizes pH and aids in renaturing of DNA strands after NaOH denatures DNA into single strands. So, logically, I am assuming vinegar should not cause denaturation.

From my experience working on Moths, I completely agree with Santosh Singh. It is very essential. It was proven right not just for my work but also for my colleague working on Nematoda.

A few factors that effected my DNA barcoding efforts were exposure to ethyl acetate and heat treatment. Sample with strong exposure to ethyl acetate (drenched like you need to dry them out or you have kept them in the killing jar for long) had more denatured DNA with very low success rate. But samples transferred immediately after killing reduced exposure and had higher success rates. Unfortunately, smaller (size) species were highly affected. Uncontrolled Heat drying is detrimental and is positively a big "NO" for specimen destined for phylogenetic workouts. You may be surprised to know, specimen dried at room temperature decades back were successful as opposed to my heat dried few months old samples.

Lastly, a friendly suggestion, to avoid breaking/ fragility of your specimen during pinning you can rehydrate the specimen using a desiccator before pinning . If you don't find one simply use some mild damp tissue papers (or plain white cloths) layer them in an air tight container. Place your specimen between the layers and give them a stand time of min. 4-5 hours (in warm weather use refrigerator to avoid degradation/foul smell). Both processes will reduce the stiffness and you will be able to work on them at ease. Also, in my personal experience I have observed that field preservation of coleopterans in 70-80% ethanol as opposed to absolute works better. I used to have minimal issues with brittleness or rigidity of the samples and rarely required rehydration for taxa as Coleoptera, Hemiptera, Blattodea, Orthoptera to mention few.

Wish this helps.

Best Regards.

Aditi Shere-Kharwar, Kamalika Bhattacharyya thank you both for your great answers!

Kamalika Bhattacharyya as far as vinegar is concerned, we routinely use 9% white vinegar (though I'm not sure what exactly "9%" stands for) for storing almost all of our beetle specimens and it works great, beetles stay whole and flexible for years, even if stored at room temperature. This also keeps them in good condition for pinning. But my colleagues told me from experience that PCR success with such (unpinned, stored exclusively in white vinegar) specimens is poor - although it might not even be the vinegar but the age of used specimens. That is why I am looking for a reliable alternative. I have considered 80% ethanol as well but ethyl acetate is very light and convenient for collecting on foot.

So if I try to sum up the discussion, ethyl acetate should be fine, but it's time and storing conditions (storing agent and temperature in the containers, drying temperature for pinned specimens) that most affect PCR success.

In my case I plan to use ethyl acetate just for killing and as soon as I arrive in the lab or at home, I'll remove a leg for DNA and freeze it in ethanol and store the rest of the beetle in white vinegar where it will remain nice and soft until I can pin it.

Matic Gabor

Thanks for the summarization.

In simple words aqueous solution of acetic acid is known as Vinegar. So, 9% white vinegar denotes the dilution level of acetic acid in water. i.e., composition of 100ml of your white vinegar is 9ml (9%) acetic acid (concentrated) and 91ml (91%) distilled water by volume.

Vinegar has a pH of 2.4 - 3.3 which is pretty acidic but should not effect DNA (threshold pH 7.0). So try to check pH level of your solution as a potential factor responsible for poor sample quality on long term exposure to the solution. Checkout the following article to see if acidity of your solution is effecting genetic material.

UHLENHOPP, E., KRASNA, A. Denaturation of DNA at pH 7.0 by Acid and Alkali. Nature 223, 1267–1269 (1969). https://doi.org/10.1038/2231267a0.

Best of Luck!!