Dear all, A few weeks ago, I sent a mail about the optimization of long-term preservation of leaf material in silica gel (see my mail below). I�ve got several answers and thank all people you provided advice and suggestions.The answers were very diverse and difficult to synthesise. Therefore, I paste them below, for other Evoldir members who might be interested. Of course, if some of you have further comments/advice, I�m still very interested in your experience and opinion! -------------- My mail: Dear all, I would like to ask for advice/suggestions about the long-term preservation of leaf material in silica gel (for molecular studies). We have quite abundant collections from worldwide field trips and wish to optimize the storage conditions for long-term preservation while simultaneously keeping this material available (easy to sample) for current research projects.To our knowledge, no real data are available yet on this topic, and we would like to know what the experience of diverse people might suggest about: - Temperature of conservation: -20�C, 4�C, or room temperature? -20�C could appear the best solution, but what about the condensation that will form on the samples when taking them out of the freezer to sample a piece of leaf? Are high temperatures (30-35�C) detrimental to DNA preservation? - Is light to be strictly avoided for DNA preservation? - Bags, tubes, boxes? Although tubes with screw-caps may be optimal for DNA preservation, they are very space-consuming. Therefore, we are rather thinking of using hermetically-sealed bags (e.g. Minigrip). These bags should then probably be placed in airtight plastic containers such as Tupperware boxes�.?? - Some people put the leaf material into an individual bag only after the leaf is dry. In this case, would you put silica gel in the bag? Or would a small amount of silica gel in the box containing several bags be enough? In short, any suggestions on the long-term preservation of dry leaf material for DNA studies would be greatly appreciated! Thanks to all who could supply ideas. Best regards, Myriam Gaudeul Department of Systematics & Evolution Mus�um National d�Histoire Naturelle, Paris (France) gaudeul@mnhn.fr -------------- From: jahodova@natur.cuni.cz I have quite a lot of samples stored in silica gel at a room temperature, that seemed to be OK when I used them within a couple of years from the collection. However, I have no experience with long term storage. I know some people prefer to keep their dried samples in -20, but I do not have facilities for all my samples at the moment. I suggest that the "strictness" of storage also depends on applications you are likely to use with your samples - as some are more demanding to DNA quality (such as AFLPs) then others (microsatellites, sequencing?). On some cases we have managed to use herbarium vouchers for AFLP (but not all samples tested worked though) - but I know some people use herbarium material quite regularly for microsats and/or sequencing. I prefer placing individual samples into a paper envelope/bag and then several of them (e.g. from one population/locality) into large re-sealable plastic bag with silica gel. The quality of such bag is a must - I prefer Ziploc heavy duty freezer bags. I've had a lots of problems with other bags (even from some laboratory suppliers) - that just were not airtight. So placing these bags into some airtight boxes would certainly be a good idea. Best wishes, Sarka Jahodova -------------- From: Nora.Scarcelli@manchester.ac.uk Bonjour, je n'ai pas de solution miracle mais j'ai moi aussi ete amenee a stocker des feuilles seches. Ce n'etait pas a proprement parler du long-terme puisque j'ai extrait l'ADN au bout de 6 mois mais peut etre que cela pourra etre utile. J'ai commence par seche mes feuilles directement sur le terrain avec du silica gel. La methode n'a pas l'air tres scientifique mais elle marche bien : j'ai mis chaque echantillon dans un filtre a cafe, puis une dizaine d'echantillons dans un sac congelation a zip contenant du silica gel. J'ai ensuite renouvelle le silica gel aussi souvent que necessaire. Une fois rentree au labo j'ai stocke mes echantillons dans les memes sacs congelation, a raison de 30 a 50 filtres par sac, avec un fond de silica gel dans chaque sac pour maintenir mes echantillons au sec. Le tout en chambre froide (environs 4�C). Apres 6 mois j'ai ouvert tous les sacs et les filtres a cafe pour prendre un echantillon de feuilles et extraire l'ADN. Je n'ai pas de probleme avec cet ADN, ni a l'extraction, ni en PCR (microsatellites). Deux ans apres les sacs sont toujours en chambre froide, j'ai juste change le silica gel pour maintenir mes feuilles bien au sec. Je n'ai pas eu l'occasion de me resservir de ces echantillons, donc je ne peux rien dire du maintient de la qualite sur 2ans, mais je suis optimiste :) J'ai elimine l'idee des tubes des le debut car j'avais peur que mes feuilles moisissent si elles etaient mal sechee (j'ai deja eu ce genre de mauvaise experience avec des graines auparavant). J'ai opte pour des filtres a cafe car ils sont poreux et permettent donc de maintenir les feuilles tout le temps au sec si on prend la peine de renouveller le silica gel de temps en temps. Pour la temperature de stockage j'ai choisi 4�C car je voulais eviter les congelation/decongelations a chaque prelevements qui auraient pu abimer l'ADN. En esperant que cela ai pu etre utile. Nora -------------- From: toneattof@yahoo.it About long term preservation of leaf matherial: I've collected leaf matherial 2 years ago, and i stored them in sealed plastic bags with silica gel. They were at room temperature, light was avoided. I've extracted the DNA recently with a CTAB method and i did not experience problems. Regards Fiorello -------------- From: mzavodna@purdue.edu I had leaf material dried and stored in silica gel for about 3 years and it worked fine for DNA extractions. I do not have experience for longer-term preservation but I suppose it would work fine. We had leaves in coffee filters (to separate and label individual leaves) and several filters in a zip-lock bag or tupperware box with silica gel. We kept them on room temperature (temperate climate), avoiding direct sun and regularly checked. The leaves were originally collected in tropics and brought to temperate European climate. Once they were dried, silica gel was hardly needed to be changed over that period. Leaves were then easy to grind and we typically used CTAB DNA extraction protocol. An alternative for leaf material preservation might be freeze-vacumm-dried material which can be kept at -20C or -80C then. I have kept material that way too and it worked fine for about the same time period as above. I am afraid, I did not need to work with that material later, so I do not know whether it was still fine for much longer than a couple of years, though I think it would be. Hope this is of some help. best wishes, Monika -------------- From: huffordk@uci.edu Dear Dr. Gaudeul, I have collected leaf samples in tropical and temperate regions and I've always preserved samples in silica gel inside ziplock bags. I've usually extracted DNA from these samples within two years of their collection with considerable success. In my collections, the key to DNA preservation is the initial quality of the leaf tissue, and the dry-down time in the bag. (It's best to start with a great deal of silica gel per sample to hasten drying; some gel can be removed once the sample is dry to reduce sample weight for shipping.) K. Hufford, UC Irvine -------------- From: gmay@umn.edu Hi, We have experience with Si gel preservation, although mostly for fungal materials. It works well for us and is inexpensive. * We store at either -20 or -80. It doesn't seem to make any difference and -20 is cheaper. * Condensation - this problem can be alleviated by _strict_ instructions to users to allow the material to come to room temperature before opening. If you are working in a humid environment, you can make a chamber with Drier-ite in which you put the tubes out of the freezer. Then open. Otherwise, yes, condensation can ruin the materials. * We store in microtubes with screw caps and these work well for us, but for bulkier plant material, your idea of sealed bags should work well. We find it useful to then organize the material in boxes with big labels on the side that are readable in the freezer. * I would find some way of putting the Si gel in each sealed bag because then each is unit. We made our own packets with drier-ite - with the color indicator so we could tell when to replace or re-dry. Maybe you could find a cheap source of the type used commercially - those small paper pouches with Si gel that seem to come in everything from cereal to computers? Hope this helps, Georgiana May, U. Minnesota -------------- From: jbrunet@wisc.edu Dear Myriam, In my laboratory we freeze dry the samples after collection and keep them in a refrigerator. Freeze drying (lyophilizing gets rid of water thus fungi will not get into your samples). -------------- From: guanacosoup@gmail.com Hi Myriam, Our current approach at the Botanic Gardens Sydney is to put dried leaves into regular paper envelopes. The envelopes are then stored inside plastic boxes with silica in the bottom and the boxes are kept at -20C. The envelopes are very cheap and are very easy to store. Just make sure your containers are air-tight or close to air-tight so that the silica gel won't require replacement too often. Best wishes, Michael Whitehead -------------- From: kym.ottewell@adelaide.edu.au Hi Myriam The only experience I've had with long-term storage of samples is with silica dried material stored at -20. I was able to get good DNA from this material ~4 years after it was preserved. The leaves were stored in small paper envelopes in a tupperware container with a layer of silica on the bottom. Otherwise for short to medium term storage I just put my samples in individual zip-lock bags in a -80 freezer. Cheers Kym -------------- gaudeul@mnhn.fr