RAPID SMALL SCALE ISOLATION OF DROSOPHILA DNA AND RNA from http://fruitfly4.aecom.yu.edu/labmanual/62.html

DNA:
isolation of Drosophila DNA; DNA isolation;
The following protocol describes the isolation of DNA from adult flies; but can be used equally well to extract DNA from other developmental stages. DNA prepared according to that method is readily digested by restriction enzymes and has an average size of 40-60 kb. In order to remove RNA contaminating the DNA preparations DNase free RNase should be included when digesting with restriction enzymes.

1) Anesthesize flies with CO2 or ether and put 1-20 flies in an eppendorf tube. Keep on ice until next step.

2) Add solution A containing 0.1 M Tris-HCl, pH 9.0; 0.1 M EDTA; 1% SDS and 0.5-1% DEPC (added directly before use) and homogenize gently with a 3 mm diameter glass or metal rod . Use 100 µl of solution A for extracting DNA from 1-5 flies, 200 µl for 6-10 flies and 500 µl for up to 50 flies. Incubate for 20-30 minutes at 70°C.

3) Add 14 µl of 8 M potassium acetate for each 100 µl homogenate and leave on ice for 30 minutes.

4) Spin in the eppendorf centrifuge at 4°C for 15 minutes. Transfer the supernatant into a fresh eppendorf tube being careful not to disturb the pellet. If you get flakes in the supernatant respin.

5) Precipitate DNA by adding 0.5 volumes of isopropanol at room temperature and spin for 5 minutes at RT. Wash the pellet carefully with 70% EtOH, respin, dry and redissolve in 10 (1 fly) to 100 (50 flies) µl TE.

RNA:
RNA isolation; isolation of Drosophila RNA;
This is a modification of the method of Chirgwin et al. (Biochemistry 18, 5294-5299, 1979) which can be used for the rapid extraction of total RNA form all developmental stages of Drosophila.

1) Homogenize tissue (e.g. 1 fly or 50-100 embryos in 50 µl and up to 200 flies in 1 ml) GHCl buffer (7.5 M guanidium hydrochloride; 0.025 M NaOAc, pH 7.0; 5 mM DTT) + 0.5% N-laurylsarcosinate + 0.5% DEPC.

2) Extract once with an equal volume of phenol-chlorororm and separate phases by centrifugation.

3) Transfer the aqueous (upper) phase into a new tube and precipitate the RNA by adding 1 µl 1 M acetic acid and 25 µl ethanol for each 50 µl of GHCl-solution. Leave at -20°C for 3-24 hours. Pellet the RNA by centrifugation for 5 min., 8-12K.

4) Remove supernatant as complete as possible and redissolve the pellet in half of the original volume (but minimally 50 µl) GHCl buffer.

5) Reprecipitate the RNA as in step 3. (The reprecipitation serves to remove DNA which will not precipitate under these conditions).

5a) optional: reprecipitate the RNA for 1-2 more times by repeating steps no. 3 and 4. This is only necessary if large amounts of DNA have to be removed, e.g. when analysing the transient expression of DNA injected into embryos.

6) Wash (and store) the RNA pellet in 100% ethanol, room temperature. For Northern analysis the RNA is best dissolved directly in Northern sample buffer (e.g. 50% formamide, 2.2 M formaldehyde, 1x MOPS).

Link: Good Quality Drosophila Genomic DNA Extraction

There is, of course, also the less specific ‘extract DNA from anything‘ kitchen method which would probably work for Drosophila as well. I’ll give it a go soon…

“I was much struck how entirely vague and arbitrary is the distinction between species and varieties”
Charles Darwin. Origen of the Species.

Its usually important to start from the basics, so this is an overview of what we mean by the various taxonomic ranks such as family, genus, species, subspecies, strain, phenotype, etc etc – the various ways in which we distinguish ‘this’ organism from ‘that one’.

Family
Family is actually quite difficult to define specifically, mostly referring to a group of related organisms within a particular order. Usually they will be grouped into a family because of obviously shared characteristics. For example, humans belong to the order of primates (primatae) along with chimps, monkeys and lemurs. We are in the family of hominids (hominidae) due to our shared characteristics with chimps, gorillas and orang utans of being able to walk more or less upright, having no tail, similar dental structure and so on.

Drosophila belong to the family of Drosophilidae within the order of Diptera (generally speaking, flies with 2 wings).

Genus
Once again there is dispute about exactly what is meant by genus. The genus will generally be used as the principle scientific name (in binomial nomenclature) for the organism – such as Drosophila for fruit flies, Canis for dogs and their relatives and Homo for humans.

According to F B Gill the rules of thumb for delimiting a genus are:
a) monophyly – all descendants of an ancestral taxon are grouped together;
b) reasonable compactness – a genus should not be expanded needlessly; and
c) distinctness – in regards of evolutionarily relevant criteria, i.e. ecology, morphology, or biogeography

Species
One of the easiest ways of defining a species within a Genus is that the organisms are capable of breeding and producing fertile offspring. In addition other factors such as similarity of DNA, ecological niche and morphology are also taken into account. Species within a genus will have the same ancestor.

Many different kinds of species have been described, each with their own criteria for defining the term ‘species’ – for example; ecological species, Biological / reproductive species, Biological / Isolation species, Genetic species, Morphological species and so on.

Some biologists may view species as statistical phenomena, as opposed to the traditional idea, with a species seen as a class of organisms. In that case, a species is defined as a separately evolving lineage that forms a single gene pool. Darwin concluded that the term ‘species’ is provisionally useful for naming groups of interacting individuals.

The most commonly species of the Genus Drosophila, and the one I am working with is Drosophila melanogaster. The species name, as well as often the genus name, will often have quite poetic roots – in our case ‘the dark bellied dew lover’.

Subspecies
A species can have none or two or more subspecies but never just one. Organisms that belong to different subspecies of the same species are capable of interbreeding and producing fertile offspring, but they often do not interbreed in nature due to geographic isolation or other factors. The differences between subspecies are usually less distinct than the differences between species.

Phenotype
A phenotype is an organism’s observable characteristics or traits: such as its morphology, development, biochemical or physiological properties and behavior. Phenotypes result from the expression of an organism’s genes as well as the influence of environmental factors and the interactions between the two. Over the course of time a species may modify its phenotype to succeed in or exploit their environment. All phenotypes of a particular organism will belong to the same species. Phenotypic variation is caused by underlying heritable genetic variation and is a fundamental prerequisite for evolution by natural selection.

Strain
A strain is a genetic variant or subtype of an organism which can be produced by inbreeding, artificial selection or genetic engineering. Strains are generally artificially produced for scientific or economical (specifically agricultural) purposes.

Decades of work preparing a miniaturized laboratory for identifying minerals on Mars have also yielded spinoff versions with diverse applications on Earth and, possibly, the moon.

This image shows one of the spinoffs, in the orange case, in use during a November 2008 expedition to the Mauna Kea volcano in Hawaii.

NASA’s Curiosity rover will carry the Chemical and Mineralogy (CheMin) instrument during the Mars Science Laboratory mission to be launched in late 2011 and investigate an intriguing area on Mars for two years. CheMin uses X-ray diffraction, a first for a mission to Mars and a more definitive method for identifying minerals than any instrument on a previous Mars mission. It supplements the diffraction measurements with X-ray fluorescence capability to gain further details about the composition of powdered rock and soil samples delivered by the rover’s robotic arm.

CheMin Principal Investigator David Blake, of the NASA Ames Research Center, Moffett Field, Calif., is seen here collecting data from a CheMin cousin called Terra. The scene is from a NASA field test of technology for producing water and oxygen from soil, using the Hawaiian site as an analog for the moon. In such an application, Terra could analyze the starting soils as well as products from the extraction process.

entire post lifted from http://mars.jpl.nasa.gov/msl/multimedia/images/?ImageID=3282

image credit: NASA/JPL-Caltech

Curiosity is a rover much bigger than anything that has been sent before, dwarfing Spirit and Opportunity and especially Sojourner. It weighs in at about 1 ton and is about the size of a small SUV. The interesting thing about this mission is that it is a definite search for signs of life – not to prove that there was water, or to look for interesting rocks, but a bona fide life hunt. Having said that, the rover carries no instruments that will detect actual life forms, instead carrying a range of instruments (that together constitute a fairly impressive mobile robotic laboratory) that will look for signs of organic matter and signs that Mars was capable of supporting organic life.

The rover carries the following range of instruments:
Cameras:
Mast Camera (Mastcam), Mars Hand Lens Imager (MAHLI), Mars Descent Imager (MARDI)

Spectrometers:
Alpha Particle X-Ray Spectrometer (APXS), Chemistry & Camera (ChemCam), Chemistry & Mineralogy X-Ray Diffraction/X-Ray Fluorescence Instrument (CheMin), Sample Analysis at Mars (SAM) Instrument Suite

Radiation Detectors:
Radiation Assessment Detector (RAD), Dynamic Albedo of Neutrons (DAN)

Environmental Sensors:
Rover Environmental Monitoring Station (REMS)

Atmospheric Sensors:
Mars Science Laboratory Entry Descent and Landing Instrument (MEDLI)

I’ll go into more detail about the specific instruments in future posts, once I’ve worked out which ones are most relevant to a ‘Proxy’ type project. Meanwhile the most comprehensive overview of the mission is obviously at NASA’s site here

Here’s a link.

I’ve recently been reading about the boggy hydrocarbon deposits which should be very common on Titan’s surface. Hydrocarbons which fall as rain will wash down the sides of the mountains taking fine rocky material with it, just as water rain does on Earth. Therefore at lower altitudes you get a hydrocarbon mud, which will closely resemble tar or thick oil. Not good for a fruit fly to land in. I’m wondering if I should include some similar material in one of the fly experiments though – the gases and smells given off by this stuff would play a significant part in the Titan experience. The drosophila would probably need to get used to it.

Much of the organic chemistry is in the form of what Carl Sagan termed ‘Tholins‘. I’ll try and write a little more about those soon.

Another chemical issue on Titan, especially as far as water is concerned, is the abundance of ammonia. Liquid water mixed with huge amounts of ammonia are regularly brought to the surface through volcanic activity. Ammonia is usually highly toxic to insects but a paper exists, published by Danial J Borash et al, which describes experiments in the evolution of ammonia tolerance in Drosophila melanogaster.

Gazing out of the window on a flight home from Trondheim last year over vast and mountainous terrain it struck me how perfect the Norwegian landscape would be for a filming and sampling location for a new edition of the installation. Fortunately Ivar at Atelier Nord and Gisle at Piksel were happy share my enthusiasm and to support me in staging a new edition of the work.

So a few days ago I went off to the far, far north of Norway and the island of Sørøya which, at almost 71 degrees longtitude, is well within the arctic circle. With expedition assistant Hillevi Munthe I made the long journey from Spain, via Paris, Oslo and Trømso to Hammerfest, and then by ferry to Akkarfjord – a village of 80 people and our arrival port on Sørøya. Final simple provisions were collected before we meet Alvin Vaseli, a teacher at the village school, who takes us in a borrowed 4×4 across the island to Gamvik, an easier starting point for the walk to the northernmost tip of the island at Tarhalsen where our cabin awaits us.

After meeting the Belgian couple who had arrived on the island 5 years previously to set up a sheep farm there (and are now branching out into some promising looking tourist accommodation) we set off on our way across the emptiness of the treeless and rugged terrain, heavy packs already cutting into shoulders. We soon realise that we have another member of the expedition – dog company in the shape of Rufus, a guest at the farm and an eager runner.

After about 3 hours of walking and resting we sight the top of the path that leads down the steep 150 meter rocky drop to the cabin. This vertiginous and winding path had been restored complete with indispensible handrails a couple of years previously by Tom Eirik Ness and Torbjorn Brataas, who we had met out marking other paths an hour or so before. The views from here, as on the rest of the island, are simply incredible. Vertical zigzags cascading down the glistening felspar, aiming towards the rocks and debris below.

Finally down at the cabin we set up a temporary home and prepare the kit for the first sample expedition. We decide to head to the very tip of the island, about 30 minutes of walking, scrambling and climbing across a series of ladders and rails, to see what kind of terrain was there, and to gain a foothold on the northernmost piece of ground. Once there it proves to be too green and grassy to be appropriate for Proxy, although it is awe inspiring to look north and imagine that there is nothing but sea from here to the North Pole, and that it isn’t so far away. The fact that the September weather on Sørøya is about 10 degrees above the seasonal norm only makes it slightly harder to believe. Returning towards the cabin an outcrop of rock a little way back down the path offers the first sample location after a short climb up the cliff face. A vial marked S01 full of dry hard moss awaits robotic analysis.

Then it’s back to the cabin and whisky and chorizo with Tom and Torbjorn, accompanied by stories and knowledge about the island and its nature. The evening is eating, resting – watching for whales and Aurora – neither of which make an appearance.

Day two is the big expedition day and totals 8 hours of walking, covering 3 or 4 peaks of 250 to 320 meters, bogs, cliffs, unbelievably strong winds, hunting for streams to drink from, amorous approaches from Rufus, filming, photographing and taking 5 samples of moss, earth and lichen. We ignore the burning, aching muscles in legs and backs and walk steeply uphill reaching Kjøttvikvarden, 12 meters of hand-piled stone from the 1850s – a landmark for fishermen. Furious winds try their best to tear us from the mountain and fling us into the sea far below. Sample 4 comes from close to the tower. Bizarre lenticular cloud formations that mimic UFOs and a vivid sunset accompany our walk and descent back towards the cabin as gulls, terns and cormorants (a sea eagle?) wheel above our heads. No whales, no sealions. A roaring fire amongst the tons and tons of driftwood on the west facing stony beach pass the evening. No Aurora. But a mink.

Day three is the final day, a day of combined sampling mission and walk back to Akkarfjord. It is also the day to try and get our friend Rufus back to where he belonged. As we approach the twin lakes of Vassvikvannet and Kjøttvikvannet we realise we have no time to walk via the amazing sandy beach near Gamvik, sand blown by fierce winds more than 100 meters up the cliff face. A vertical beach. We go via Trollbuktfjellet, the highest point on the most direct route back to Akkarfjord. Sample 7 is taken from an almost dried up small lake – reindeer tracks, bird tracks, small unidentified animal tracks mingle with my own in the mud. Sample 8 – the final one needed – is taken from a rocky outcrop just over the top of the peak. Then it is the long walk back, uphill and downhill, towards the village where only once, maybe twice, we question if we might not make the 16.45 ferry back to Hammerfest and civilization. In the end we make it with time to spare, there is time for coffee and biscuits at Alvin’s house and a solution to the Rufus situation. We join Tom and Torbjorn on the ferry back – their week of path marking and fixing finished.

All too soon it’s back to airports, people and rules and regulations. Multitudes of people and movement a shock to the system.

Sørøya is a magical place with wonderful people. I’ll be back there as soon as I can.

As far as Proxy is concerned I think I have some amazing images and footage, and I can’t wait to find out what the robots discover in the samples I have collected.

Big thanks to Alvin Vaseli, Christine Witt, Tom Eirik Ness, Torbjorn Brataas and Rufus who all contributed to the expediti0n in different ways and willingly shared their kindness, knowledge, time and companionship.

One of the main considerations for this project was that I wanted to stick to a scientifically rigorous process all the way through, while developing a project rich in artistic metaphor and poetic statements. Rigorous metaphor…

Seeing as I hadn’t worked with Drosophila before it was really useful to have Janine take the lead in the preparation of the flies. Even though 4 phenotypes were ordered, apparently you mustn’t assume that these are ‘pure’ species. (the number of times the terminology of this project gets close to eugenics is actually quite alarming). We don’t know if the females have mated with anything else before they were packaged up and sent, therefore what is necessary is separation of males from virgin females. Virgin females are procured by waiting for new flies to eclose – to emerge from the pupa. Seeing as they don’t become sexually mature for several hours we know that if we get them early enough they will be virgin.

So, all newly eclosed flies are anaesthetised using the quite nasty ‘Flynap’ and examined under the dissecting microscope. Females and males are identified and the females are placed into the new phial for virgins. It becomes fairly easy to distinguish between males and females once you get your eye in. The male has a darker patch at the rear of the abdomen and a form of plate with tiny protrusions.

After 3 or 4 days of this process we collected enough flies to have 4 male and 4 female of each phenotype for experiments and 4 male and 4 female of each phenotype for control.