The very mysterious case of the Ouessant fly internship... · 25°C, night:16-18°C) and were identified upon emergence of adults. D. Poinsot The very mysterious case of the Ouessant

The very mysterious case of

the Ouessant fly

UE Bio 818 Communication Scientifique

D. Poinsot The very mysterious case of the Ouessant fly

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Santaklaus Wildlife Institute, Aarvård University, Greenland

Lab internship position available for three month (Starting: January 1st, 2009) Bacteria-related life history traits in a new Drosophila species Context: Drosophilids are the well known fruit flies developing from decaying vegetal material, the most famous being the laboratory model Drosophila melanogaster, which is found worldwide. We have discovered a new drosophilid species (D. enezeusae) which is wingless and strictly endemic to Ouessant (Enez Eusa, in Briton), a small windy island near the French Atlantic coast. Very surprisingly, D. melanogaster itself seems almost absent from this island. Project outline: After our initial publication about the discovery of D. enezeusae, we have found that this insular population is infected by the endocellular bacterium Wolbachia. In many species, Wolbachia are known to modify life-history traits of their hosts, (such as fecundity, longevity or sex-ratio) and can lead to reproductive isolation between infected and uninfected individuals. Your project will investigate whether this is also the case in D. enezeusae. For that purpose, you will obtain uninfected strains by curing flies from their bacteria, using an antibiotic treatment (tetracycline) and observe the result of crosses between infected and uninfected individuals. Moreover, the possibility of hybridisation between D. enezeusae and D. melanogaster will be tested. Job profile: the successful applicant will have a BSc in biology and a keen interest for ecology and evolution. Previous experience with insect rearing and some basic molecular biology skills (PCR) are desirable. The ability to quickly distinguish a polar bear from a heap of snow might also be useful.

Contact Dr. Miko Nunaatuk Santaklaus Wildlife Institute, Aarvård University, 10 Harfang Lane, Aarvård, Greenland [email protected]


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Dr. Miko Nunaatuk Santaklaus Wildlife Institute, Aarvård University, 10 Harfang Lane, Aarvård, Greenland

25 miles north of Umaatuktituk, January the 7th, 2009 Dear trainee, A happy new year to you ! I am very glad that you accepted this internship position and on behalf of all the staff I wish you a very good stay at the Santaklaus Wildlife Institute (which you will very rapidly call “Santa” as everyone else). Wuk Qaamanassut, our caretaker, has certainly shown you into your room by now, and he has explained to you that the solid steel bars sealed to your window frames are not here to prevent your escape but rather to forbid rogue bears smashing their way in without being invited to. He also told you that because of those beautiful but dangerous neighbours it is strictly forbidden to wander alone on the campus after dark. Wuk also gave you a blanket for your bed. You may need it; it can get a bit cool at night.

I will have the pleasure of being your supervisor for the duration of your internship, during which you will study Wolbachia-related life history traits in D. enezeusae, as previously agreed. Unfortunately, you will have accept remote supervision, because I have just been drafted to take part in the big decennial bear census, which means that for the next three month I will be on the move counting polar bears during their yearly migration, which means that I will not have the opportunity to meet you face to face. Fortunately, I have access to my e-mails via satellite link, so we can communicate anyway. Because weather conditions are a bit rough here, the connection can be erratic, especially during aurora borealis flares, so do not despair if I do not reply your requests immediately. You may ask me any questions you would ask to your supervisor but I cannot correct the paper for your exam: I do not read French at all! Of course, do not hesitate to ask other Aarvård students for advice about everyday questions such as “The ink of my fountain pen has frozen solid! Do you know where I could buy pencils like those you use?”. I wish you a good stay up here and please... Aargåff! (:)) Cheers, Miko You can contact me anytime at [email protected]


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Proceedings of the Plougastel-Daoulas Academy of Sciences 58:1027-1031. DOI:12451.547.245 SHORT NOTE Drosophila enezeusae (sp. n.), a wingless drosophilid species

from Ouessant

Miko Nunaatuk1 & Trevor McIntosh2

1 Santaklaus Wildlife Institute, 10 Harfang Lane, Aarvård, Greenland 2 Loch Ness School of Biological Sciences, 105 Square Crescent Road, Inverness, Scotland

Abstract — Here we report the discovery of Drosophila enezeusae (sp. n.), the first apterous drosophilid species ever described, which appears to be strictly endemic to Ouessant, a small and windy oceanic island a few miles off the westernmost tip of Brittany, France. Apart from its lack of wings and smaller size, D. enezeusae is morphologically almost undistinguishable from D. melanogaster in the female sex, although D. enezeusae males are easily recognisable by their distinctive genitalia. In Ouessant, D. enezeusae is the only drosophilid we could find. Quite incredibly, D. melanogaster itself seems totally absent from the island, except for very rare individuals found near containers of fruits recently imported from the continent. The reason why such a widespread species as D. melanogaster has not yet succeeded in colonizing an island receiving so much traffic from the mainland is deeply puzzling, and the evolutionary origin of D. enezeusae is not clear.

INTRODUCTION Drosophilids (Diptera : Drosophilidae) are small fruit flies originating from the tropics, and counting among them the famous genetic’s lab workhorse Drosophila melanogaster, the first animal ever to have its genome fully sequenced. Yet, despite of a century of research and thousands of papers dealing with this animal model, surprisingly little is known about the basic ecology and behaviour of drosophilids in the field, although a large number of species have been described. The current count stands at over 3,000 (Russo et al., 1995), the most recently described species being D. Santomea, which is endemic to São Tomé island in the Gulf of Guinea and was discovered at the turn of this century (Lachaise et al., 2000). In this short note we describe the serendipitous discovery of yet another endemic species, Drosophila enezeusae (sp. n). Contrary to Drosophila santomea, which is more than 200 kilometres from the African coast, we found D. enezeusae nearly on the doorsteps of the many scientific labs housed in the university of Brest, more precisely on the small windswept French island of Ouessant, which has been inhabited for centuries and receives tourists by the thousands every summer. The present note deals with morphological characters only, the basic life history traits of the new species will be the subject of a future more ecology-oriented paper.


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MATERIAL AND METHODS The island of Ouessant

Ouessant (48° 28' 00'' N 05° 04' 60'' W in Lampaul) is a small island (4 × 7km, 1158ha) situated 12 nautical miles (approx. 20km) from the westernmost tip of Britanny, France. It enjoys a mild oceanic climate (maximum daily august temperatures: 22°C, minimal February temperatures: 6°C) with a typical rainfall of 700mm (similar to Paris). On the other hand, Ouessant is very windy, being subject to 178 days of winds exceeding Beaufort 4, including serious gale force winds in autumn and winter. As a result, the vegetation is low (trees are almost absent) and constituted essentially of a low turf near the coast and moors further inland. The island is criss-crossed by a network of low stone walls, which are not maintained as they used to be and are often hidden by brambles overgrowth (Rubus sp.).

Mammals are rare on the island, but the avian fauna is quite rich both in marine and migratory species, such as the Northern Fulmar (Fulmarus glacialis), Great Shearwater (Puffin gravis), European Storm Petrel (Hydrobates pelagicus) and raptors such as the Osprey (Pandion halietus) or the emblematic Peregrine falcon (Falco peregrinus), which is observed nearly all year long (Audevard, 2006). Finally, Ouessant is home of 960 Ouessantins in winter, and receives several thousands of visitors during the summer season. As far as the historical record shows, it has been inhabited continuously in historical times, and archaeological evidence suggests a human presence as early as 1500yrs BC. Sampling sites Ouessant being a very small island, the 13 different locations sampled cover most of the area and are representative of the vegetation cover found. Their characteristics are described in table 1.

As a second step, nearby continental locations (Brest, Plougastel-Daoulas, the Crozon Peninsula) were included in the sampling scheme, as well as the neighbouring islands of Molène, Hoëdic or Sein. However, not a single Drosophila enezeusae was captured from this second series of traps (which yielded a total of 3,428 flies of the genus Drosophila). Accordingly, this paper will only deal with the Ouessant results. Sampling method All sampling in Ouessant was carried out on 5-10 August, 2008. Plastic bottles (250ml) were fitted with a U shaped aperture on the side and baited with ripe banana and baker’s yeast. The bottles where left in place for seven consecutive days, shaded from direct sun to avoid overheating (whenever possible, they were left dangling under brambles bushes (Rubus rubus). Upon collection, each trap was immediately sealed with cellotape and adult flies seen in the bottle were captured with an insect aspirator for examination under the binocular after light diethyl-ether anaesthesia. The larvae were left to develop in the shade at ambient temperature (day:20-25°C, night:16-18°C) and were identified upon emergence of adults.


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Identification All drosophilids captured in Ouessant were all compared with the reference Drosophila collection of the first author under a dissecting microscope (max. magnification of 40x). RESULTS A total of 601 drosophilids were collected. With the exception of the trap placed in the fruits section of Lampaul’s grocery store (where two Drosophila melanogaster individuals were recovered out of 40 captures, table 1), all individuals belonged to an apterous and yet unknown species of the genus Drosophila, which we chose to name Drosophila enezeusae for its supposed endemic nature (the Breton name of Ouessant is Enez Eusa).

Table 1. Sampling sites and identification of Drosophila sp. captured in Ouessant (5-10 August 2008). Each site was fitted with a trap baited with ripe banana and baker's yeast (see text for details)

Individuals recovered Location Characteristics D. enezeusae D. melanogaster

Lampaul 1 Private garden 22 – Lampaul 2 Grocery store* 38 2 Mez Notariou Bramble patch 147 – Locqueltas Sheep pasture 50 – Nividic Point Low coastal turf 3 – Niou Uhella Old pasture 15 – Kergadou Bramble patch 153 – Calgrac'h Cove Low coastal turf – – Stiff Point Low coastal turf – – Penn Ar Lan Wetland 17 – Stang Merdy Wetland 8 – Lann ar Grac'h Moorland 12 – Penn ar Roc'h Cove Low coastal turf 4 – Feunteun Velen Bramble patch 135 – * trap left for one night only, at owner's request 599 2

Diagnosis Apart from its lack of wings, D. enezeusae is extremely similar morphologically to D. melanogaster in females (figure 1). On the other hand, D. enezeusae males are readily distinguishable from D. melanogaster males by their very conspicuous black genital plate .

A comparison of the aedeagus confirms a marked morphological difference (fig 1 d,e). These data strongly suggest that D. enezeusae is a new species, although it is obviously very closely related to D. melanogaster.

Another distinguishing character between the two species is the smaller size in D. enezeusae. (it is approximately 20% smaller than D. melanogaster in both sexes). This reduced size is not due to poor food sources since it is maintained after several generations in the lab in un-crowded conditions on high quality medium (data not shown).


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a

b

c

d e

a

b

c

d e

Figure 1. Male (a,b) and female (c) of D. enezeusae. Notice the lack of wings and the conspicuously large genital plate in the male (arrow). Pannels (d) and (e) show a comparison between the male genitalia of D. melanogaster and D. enezeusae, (respectively). Notice in D. enezeusae the very large genital plate, the broader aedeagus as well as its peculiar trident shape. Bar: 100µm.

Voucher specimens of the new species have been donated to the University of Brest. Holotypes are kept both at the British Museum (accession number BMS.251.278.1.695) and the Aarvård University Natural History Museum (accession number AAR.26.586.984) DISCUSSION

[not reproduced here so as not to stiffen your creativity in writing your own

discussion about the very mysterious case of the Ouessant fly]


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CONCLUSION AND PERSPECTIVES The discovery of a new Drosophila species brings more questions than it answers, the first being of course : is it a bona fide species or merely a subspecies of D. melanogaster? To answer it, crossing experiments with D. melanogaster will be carried out shortly. Moreover, we will determine whether the reproductive isolation between these two drosophilids is due to pre-mating (i.e. behavioural) or post-mating (genetic) factors. If D. enezeusae is, as we believe, a true species, the question of how its speciation event took place and the extremely puzzling absence of D. melanogaster on Ouessant must be investigated. Acknowledgments The first author wishes to thanks his friend B. Le Garff, whose extensive knowledge of Ouessant was a great help in discovering what D. enezeusae — if only it existed for real — might develop on: blackberries. My own initial “guesstimate” (fallen cider apples) was ridiculous since there are virtually no trees in Ouessant! The skilful hands of T. Frétey and B. Le Garff were also helpful in strategically altering the genitalia of a genuine south american drosophila species to create those of D. enezeusae. The (beautiful) original ink drawings of genitalia are due to Marta Erps Breuer (1902-1977), designer and lab technician at the Universidade de São Paulo, and a former Bauhaus student. REFERENCES Audevard A., 2006. L'année ornithologique. Bulletin Ornithologique Île Ouessant 15:1–

56. [http://audevard.aurelien.free/IMG/pdf/Bulletin-ornitho_Ouessant_2006.pdf]

Lachaise D., Harry M., Solignac M., Lemeunier F., Bénassi V & M.L. Cariou, 2000. Evolutionary novelties in islands: Drosophila santomea, a new melanogaster sister species from São Tomé. Proceedings of the Royal Society of London B. 267:1487–1495. doi:10.1098/rspb.2000.1169

Russo, C.A.M., Takezaki N. & M. Nei, 1995. Molecular phylogeny and divergence times of Drosophilid species. Molecular Biology and Evolution 12:391–404.


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Useful information to get a feel of your experiments. Basic drosophila lab equipment Drosophila bottles Such bottles are typically 250 ml flasks with a wide aperture for easier manipulation. They are filled with a one-inch layer of drosophila medium (25ml). The traditional (and most practical) way to close drosophila bottles is by means of a large cotton wool plug, which can be removed using the little finger alone if necessary, the rest of the hand being free to manipulate various objects. For better hand comfort, crude (non hydrophobic) cotton wool is used. Drosophila tubes These vials are typically not more than 15cm high (i.e., short enough to be put upright under a binocular) with a 2,5cm or wider diameter (i.e. a wide aperture). They have a flat bottom so that they can safely stand upright on their own, although they are moved around in bunches of 20 using plastic vial holders. Each tube holds a one inch layer of drosophila medium (5ml). Again, the most practical way to close them is by means of a small crude cotton ball. Drosophila medium The medium is the solid food seen at the bottom of bottles and tubes found in all drosophila labs. Since the days of Thomas Hunt Morgan (who used... ripe bananas) several dozens of media have been invented, but the basic components are usually wheat or maize flour and baker's yeast, cooked together with agar to form a solid layer firmly attached to the bottle, which can be put upside down and bumped on a rubber mat repeatedly without any medium falling. Some media use live bakers' yeast, others are axenic (sterile, at least at the moment when they are put into the bottle). David's medium (see further below) is an axenic medium. Rubber mats Also known as « bottom bumpers », they are small rugs (15x15cm) made from any soft and bumpy surface (often odd bits of used carpets) which are put on drosophila lab benches so that the bottom of drosophila bottles or tubes can be briskly bumped on the bench without breaking. This fast bumping action is one of the most frequent gesture seen in drosophila labs around the world. Its role is to make all flies fall temporarily at the bottom of the bottle so that the plug can be open without any drosophila escaping. This gesture is used in particular when flies need to be mass-transferred from an old bottle into a new one to maintain stocks. Insect "pooters" They are small « fly aspirators » made of one straight glass tube with an internal diameter of a few millimetres (enough for a drosophila to be sucked


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in) fitted tightly by a long plastic tube which the manipulator holds in his/her mouth. This aspirator is used to suck a fly in or flush it out, allowing it to be transferred from one tube to the other without the need to open the cotton plug (the glass tube is small enough to be inserted while the plug is in place). Needless to say, the junction between the two tubes is fitted with a fine fly-proof screen; otherwise drosophila workers would eat a large number of flies every day. Etherizers They are the traditional devices used to anaesthetize (or kill) drosophila. They are made of a chamber where ether diffuses and into which flies fall through a funnel. The difference between ether fly anaesthesia and ether fly murder being only a subtle question of timing, which strongly depends on the amount of ether put into the apparatus, the temperature of the room and the experience of the scientist, beginners are guaranteed to involuntarily slaughter quite a number of innocent flies before they get used to their etherizer. In modern labs, etherizers tend to be replaced by CO2 flow beds, but these have a rough surface and when it comes to sort rapidly large numbers of flies, they are not as efficient as the traditional ceramic white tile on which you dump your anaesthetised (or dead) flies. Fly strains in our laboratory D. enezeusae (wild type) Our D. enezeusae lab strain was established in August 2008 from more than a hundred females recovered captured from traps baited with ripe banana and yeast put in various places on the island of Ouessant during my summer vacation in France (for a full list of locations see table 1 in Nunaatuk & Mc Intosh, 2008). Since that time, the strain (known in our lab as « Ouessant 07 ») is mass-bred here in the entomology lab of the Santaklaus Wildlife Institute. The strain is maintained on David's axenic medium (David J., 1962. A new medium for rearing Drosophila in axenic conditions. Drosophila Information Service 93:28) with at least 400 reproductive couples per generation to minimize drift and inbreeding. We have found by PCR that all flies from this population are infected with a newly described variant of the endocellular bacterium Wolbachia,. Accordingly, during your experiments, those flies will be identified (on bottles, tubes etc.) as « w ». D. enezeusae (wolbachia-free) Aposymbiotic (i.e. wolbachia-free) D. enezeusae will be obtained from the wild type stock by letting 200 inseminated females lay for 8h on a bottle containing 25ml of David's axenic medium with 0.1% tetracycline (weight/volume) following the protocol described in Hoffmann et al. 1986 (Hoffmann A.A., Turelli M. & G.M. Simmons, 1986. Unidirectional incompatibility in natural populations of Drosophila simulans, Evolution 40:692-701). This antibiotic kills Wolbachia during the development of the larvae which feed on the medium. You will have to carry-out this treatment immediately upon your arrival in our lab, and experiment 1 (see protocols)


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will be carried out on flies emerging from the medium supplemented with tetracycline. This strain will be identified in our lab as « Ouessant 07 a », but for convenience, during your experiments you will identify it (on bottles, tubes etc.) simply as « a ». Summary :

Table 1. Strains to be used during the project

Identification Species Full strain name Infection status

w D. enezeusae Ouessant 07 infected

a D. enezeusae Ouessant 07 a uninfected

Methods for crosses Traits measured: (i) fecundity, (ii) hatch rate, (iii) sex-ratio. D. enezeusae crosses (males × female): w×a, w×w, a×w, a×a 1. Upon arriving in the lab in the morning, as early as possible after the beginning of the photophase in the 25°C climatic chamber, choose a drosophila bottle from the lab culture where flies are only starting to emerge. This bottle will have been primed at the previous generation by allowing 50 couples of flies to lay eggs for 8h only (on 25ml of David's axenic medium), to prevent overcrowding of the culture.

2. Remove the flies already present in the bottle (upturn the bottle over an etherizer and tap it down to make the flies fall in the etherizer) and throw them away in the fly graveyard (any large aperture container half-filled with oil).

3. Approximately 6h later, remove the flies which hatched since the morning, but this time do not kill them, only anaesthetise them slightly with the etherizer, then dump them on a white ceramic tile and rapidly separate males from females under the binocular, using a fine paintbrush. Males are readily recognised by their conspicuous protruding dark brown genital pieces. Gather at least thirty virgin males and as many virgin females (our preliminary observations show that D. enezeusae do not mate until at least 8h after emergence).

4. Put the females in one plugged tube (containing 5ml David's axenic medium) and the males in another (since males emerge 12-24h before the females, you may need to use more than one bottle from the culture to get enough individuals in a single day). Be careful that anaesthetized individuals do not get stuck to the medium: put them gently on the side of the tube and put the tube upright only when all flies are mobile again.

5. Let the flies mature for another 24h à 25°C.

6. Next day (i.e. 24h later), take the flies one by one using an insect pooter and form couples (one male, one female), which you will put each in one in a tube with 5ml of fresh David's axenic medium. Beforehand, scratch the surface of the medium with the tip of a lancet to form small grooves and thus stimulate egg-laying (flies do not like smooth surfaces, they prefer to lay eggs in small crevices).


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Identify the male and female of each cross by marking the tube with a waterproof felt tip marker: strain (W or A) + individual number.

7. Then, continuously observe the couples for one hour (arrange them in a half circle on your bench and scan them continuously). Do not worry; you can not overlook copulations when they occur since sperm transfer in Drosophila takes several minutes. Make a note of which couples are indeed copulating.

8. After one hour, discard the couples which refused to mate and remove the males from the other couples using an insect pooter.

9. Put the tubes containing inseminated females back at 25°C.

10. After 24h, discard the females and count the eggs in each tube under the binocular. Drosophila eggs are easy to see thanks to their two « horns », even when they are inserted in a crevice. This will yield the fecundity data.

11. Let the tubes at 25°C for another 24h to allow the eggs to hatch.

12. The next day, count in each tube the eggs which have not hatched (hatched eggs appear as flat, wrinkled and transparent chorions while unhatched eggs are white and turgescent). Any egg that has not hatched at this time will be considered as dead. By reference to the fecundity results obtained previously, calculate by difference the hatch rate (i.e. percentage of eggs that did hatch).

13. Put the tubes back at 25°C until the end of the development of the flies (approx 9 days at 25°C)

14. From the 9th to the 11th day, tap the unplugged tube each day over the funnel of an etherizer, count the adults emerged and sort them by sex. After 11 days, emergence is considered to be complete. You can then calculate the sex-ratio (expressed here as the percentage females). Methods for PCR verification of infection status

Variable measured: presence/absence of Wolbachia by amplification of gene wosp using speficic primers, the full method is described in Braig et al., 1998 (Braig H.R., Zhou W. Dobson S.L. & S.L. O'Neill, 1998. Cloning and Characterization of a Gene Encoding the Major Surface Protein of the Bacterial Endosymbiont Wolbachia pipientis, Journal of Bacteriology 180: 2373-2378).

DNA extraction. 1. Etherise 10 flies picked up at random and put them individually for 30 min in the freezer (-18°C) in a dry 1.5ml Eppendorf .

2. put on sterile gloves

3. pipet 500µl of sterile ringer in a sterile 1.5ml Eppendorf.

4. put the fly on a sterile glass plate. Using sterile fine needles, separate the abdomen from the thorax and put it in the ringer.

5. crush crudely with a sterile disposable nib.

6. add 10 units of proteinase K, close the Eppendorf and incubate 30 min at 37°C.

7. with a fine needle, prick a hole into the cap of the Eppendorf so that it does not


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pop open during next step

8. put the Eppendorf in holes on a polystyrene raft and float for 5 min on boiling water to inactivate proteinase K.

9. spin for 5 min at 10 000g.

10. pipet 10µl of the supernatant and put into a new Eppendorf which you will identify with a fine permanent felt-tip pen.

11. use immediately for PCR or store at –20°C. PCR protocol 1. Prepare the equipment : 100 microlitre PCR Eppendorfs, special PCR pipetman cones, Crushed ice in a polystyrene box, mineral oil, bidistillated sterile water, Taq buffer, dNTP oligomeres, primers 335F = 3' ACG TGG ACC TGA GGG GAG 5' and 526K = 5' TGA AGT TTG GCG TAG GCG 3' , Taq polymerase, DNA samples, benchtop centrifuge, PCR cycling machine, positive control (DNA from an infected fly), negative control (DNA from a non infected fly), DNA-free control.

2. put sterile gloves on.

3. put on ice the buffer, MgCl2, dNTP oligomeres, primers, Taq and DNA samples.

4. In an Eppendorf prepare the "premix" by mixing successively, in that order, 1020µl bidistilled water, 340µl buffer, 136µl of dNTP oligomers dNTP, 68µl of each primer and finally, 68 units of Taq polymerase.

5. close the premix tube and shake vigorously for a few seconds, then centrifuge briefly in the benchtop centrifuge

6. prepare 49µl Aliquots in each PCR tube. you will then obtain the following concentrations : 200µM of each dNTP, 0,5µg/µl of each primer and 2 Taq units per tube.

7. add DNA samples (one per tube, 1µl, obviously change the Pipetman tip each time !). The three last tubes will receive the positive control, the negative control and the DNA-free control.

8. Cover each sample with one drop of mineral oil, which will prevent evaporation during PCR.

9. set the cycling machine on 98°C for 3 min then 28 cycles [30s at 94°C, 30s at 52°C, 1min at 72°C] and final elongating period of 7 min à 72°C. Preparation of the agarose gel. This protocol is standard and can be found in any textbook 1. Weight 1.5g agarose and pour in a 250ml Erlen.

2. add 100ml of TBE 1X buffer to get a 1.5% gel.

3. boil until dilution

4. pour the gel on an electrophoresis plate which ends you will have obliterated with cellotape. Do not forget to immerse the combs which will create the wells in your gel, otherwise you will end up with a block of agarose which will be completely useless !


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5. When the gel is set, delicately remove the combs.

6. Immerse in the TBE bath.

7. pipet 10µl of each PCR product and put each in an Eppendorff. Add to each 1µl of colour marker in glycerol 8. load each well with 10µl of coloured amplification product. Take care not to damage the sides of the well with the tip of your cone.

9. do not forget to load the Kb ladder (size control) in one well

10. migrate for 20 minutes at 100V

11 reveal by bathing for 15 minutes in BET. Warning, BET is dangerous.

12. reveal the gel under UV and take a picture or scan for future reference.

Results and statistical analysis : Will be available in details shortly in your personal dataset file at http://perso.univ-rennes1.fr/denis.poinsot)

--- Good Work ! ---

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The very mysterious case of the Ouessant fly internship... · 25°C, night:16-18°C) and were identified upon emergence of adults. D. Poinsot The very mysterious case of the Ouessant