Ten simple rules to make the most out of your undergraduate research career
WHEN FIRST JOINING THE LAB:
These readings are essential!!!! Please read. They are also worth re-reading several months after you have joined the lab, as many things which initially made little sense, will make a lot more.
For most of our flypushing (i.e. collecting flies of specific genotypes and sex, setting up crosses, etc) we use a shared flylab space (room 503 LSB) with the Jacobs lab that primarily use Drosophila. The Jacobs lab also has expertise in fly genetics and molecular genetics (and protocols like immunohistochemistry). As we use a shared room we have a common set of rules and protocols to keep life in the room happy and healthy for both flies and humans. The link is here.
We have a number of useful resources for getting started with using flies. The books are all available in the lab, and for a few we have a lab PDF in the shared dropbox folder (please contact me for access if you do not already have some).
Please read the excellent How to design a genetic mating scheme: a basic training package for Drosophila genetics. The full set of downloads for this (including some powerpoints and exercises) are here. The article describing the overview of this package can be read here
here are some useful things to read:
Hard copy books: Fly Pushing (Greenspan): Chapters 1, 4-5
Drosophila: A Practical Approach (Roberts, ed.): Chapter 1 (also chapter 9 for behavioural work).
Drosophila Guide (Demerec): p. 3-16, *30-44 *optional/use as reference
Drosophila Protocols: chapter 35
Developmental Biology: A guide for experimental study (Tyler). chapter 8 (in particular, pages 94-101)
We also have many online resources in the lab dropbox folder (DrosophilaTrainingTutorials). This includes papers outlining important techniques like the UAS-GAL4 system, etc..
The Atlas of Drosophila Morphology (a PDF is available on the lab dropbox account) is a great way of learning the major morphological markers of flies.
A clearing house for Drosophila teaching and genetics resources
Definitely also take a look at the Drosophila Information Service which is an annual volume of short research, technique and teaching notes. This goes back almost 100 years with lots of cool stuff and it is a great place to find protocols that may be related to something you plan to do.
At the Bench: Chapters 1, 3-9. (Everyone should read these chapters).
-If you are doing DNA/RNA work then also read Chapters 12,14,15.
-If you are doing microscopy read chapter 16 of at the Bench. (Also read Chapter 3 of "Developmental Biology: A Guide for Experimental Study").
If you are doing any molecular cloning read chapter 11.
Further reading will be required for specific projects.
- Get fly food out of fridge. ~1 tray of bottles and 1 of vials (tegosept and non-tegosept).
- Get brushes, funnels, transfer pads, paper towel, eppendorf tubes and yeast from freezer
- Put fly food in fridge, properly bagged
- Put brushes, funnels, transfer (bunging) foam pads, paper towel, eppendorf tubes and yeast in freezer. This prevents any contamination of food mites.
- Make sure gas is off. (clear pressure from system)
- Turn off lights
- NO MASCARA!!! This gets on the microscope oculars and is very hard to clean off!!!
- Do not turn light source above 70 as it will increase chances of burning out a bulb
- If you will be working with multiple lines, clean plate and switch brush between lines
- When finished, spray CO2 plate, brush, and surrounding area with 70% ethanol and wipe, Cover. You can also disinfect with 5% bleach (but rinse with water after). This prevents infection from bacteria and molds and will kill stray flies, eggs or larvae.
- Check with others and turn off gas if not in use (clear pressure from system)
- Keep vials and bottles covered (flugs should be in). Otherwise random flies will get in and lay eggs (contamination!).
Maintain awareness of your neighbors and collaborators, and any flies they may be contributing to the local environment. Diligently hunt down any easily caught flies with a paper towel (or your finger).
Check ~every 3 generations for appropriate eye colour markers, wing markers., balancer, etc. to make sure there is no contamination from other fly lines or mites. There is no shame in something happening, there is much shame in not reporting it! If you are not familiar with what the markers should look like (we do have a poster on the wall), we have a copy of the Atlas of Drosophila morphology on the lab Dropbox account, so ask me for access.
When vials are down to about half a tray, get another from the fridge to warm up. If food in fridge is running low, do a quick inventory of how much we have and how much you/others will be using. We will be making food weekly, but this needs to be organized.
If a large pile begins to accumulate, take them when going to get food and put in the Lab freezer for a minimum of 48 hours. Move to fly food room (503 LSB) the next time you are over there. Keep the room tidy!
1- When working with KEY strains like ORE and SAM (and derivatives) only work with one strain at a time at the bench. That is do all of your work with the SAM strain (i.e. virgin collections, crosses, adding water, etc...), and then clean off your CO2 plate, get a fresh brush, etc... just as if you were finishing all of your fly work. Put these stocks away, and then, get the other stocks (i.e. ORE), and proceed with the work for them. You should not have the ORE and SAM strains in physical proximity while you are working, so that there is no chance of accidentally grabbing the wrong strain for use.
2- Pre-label vials, and CHECK and then RE-CHECK LABELS as you are working. This is the most common cause of contamination. During routine transfers of flies to fresh vials, two strains are placed in the wrong position in the tray, and then when they are being used for crosses the labels are not checked carefully enough and the wrong flies are used. You may wish to re-organize your stocks so that it will be less likely for you to make a mix up (i.e. do not keep two strains that are morphologically indistinguishable next to one another), or at the very least easier to diagnose when a mix-up or contamination has occurred.
3- NEVER HAVE MULTIPLE GENOTYPES ON THE CO2 PLATE AT THE SAME TIME!!!! Despite it's apparent efficiency, never have a mix of strains on the plate at the same time. In addition it is best to keep the vials/bottles for the vials on your place in the same spot as you work, and then move them out of reach as you start to work with the next set of strains.
4- When looking at progeny of crosses or from your stocks that appear "unexpected" in some way (i.e. wrong phenotype, white eyes when they should be red or vise-versa.) please come speak to me or at the very least show it to other people in the lab to help identify if it is new and interesting for a cool reason or simply a result of contamination.
5- Report any possible sources of contamination to me immediately. I will not be upset if we catch it early, I will likely be much less pleased if it is left un-checked, and spreads.
6- If a fly falls off of the CO2 plate while you are working, DO NOT USE IT. Treat it as a source of contamination. Similarly if there is a fly on the plate that looks wrong (i.e. it is off on one corner of the plate), assume that it is a source of contamination and remove it immediately. It is also important to check the CO2 plate for any stray flies that may have landed on the plate, or did not fall into the morgue BEFORE putting a new set of flies on the plate.
7- Organize yourself well before setting up mass crosses. When you are performing a lot of crosses all at once (say more than 10), the possibility of contamination can increase very quickly. It is very important that you organize everything very well before you start the fly work (pre-labeling vials, like with a molecular experiment). Again, it is EXTREMELY IMPORTANT to check and re-check labels, and the flies you are working with because it is very easy to get strains mixed up when you are working with a lot of them. Develop some protocols to double-check your work.
8- If you think you may have accidentally mixed up lines in a cross, or mis-labeled a strain, write a note on the vial/bottle (not just in your lab notebook), and report it to me and we will figure out a way of checking it. If it is a cross that can be redone easily, throw the questionable one out, and redo it.
9- Double check with Ian prior to completely throwing out any main fly stocks
- In general when setting up a new vial you should sprinkle some live yeast (in dispenser in freezer) onto the surface of the food. If you are have just a few flies in the vial (2-5) one or two granules is enough. If there are 10+ then you can be more liberal about adding it.
- At Room Temp, vials should be transferred no later than 18 days after setting up. Parents can be removed once larvae are present (~3-5 days). Can wait longer if desired, no later than ~9 days.
- At 24 degrees, vials should be transferred no later than 16 days after setting up. Parents can be removed once larvae are present (~3-5 days). Can wait longer if desired, no later than ~8 days.
- At 18 degrees, vials should be transferred no later than 21 days after setting up. Parents can be removed once larvae are present (~5 days). Can wait longer if desired, no later than ~10 days.
- When maintaining stocks, keep at least one copy as back up. Between the copies of a line, transfers should be staggered; transfer only if larvae are present in the copy. Ideally, both copies should be checked at once- when transferring flies from one copy, dump parents from the other
- When transferring, check and add yeast, water or paper towel as needed.
- Label vials or tray with the date transferred to help yourself remember when they were last looked at/when they need to be transferred next.
Drosophila are susceptible to numerous diseases, but are most commonly plagued by overgrowth of yeasts, molds and bactiera.
For bacteria: In particular we have some very serious issues with some Acinetobacter species that are highly antibiotic resistance. For this we have some special protocols that can be read here.
For molds: the mold inhibitors in the food usually keeps the molds at bay. If you start seeing some molds (in particular a very green coloured one), transfer the adults to a fresh vial and do several rapid transfers to fresh food. I usually do it 2-3 times on the first day I notice it (once an hour), then 1-2 a day for the next 3 days. If there are larvae in the vial (and it is a very precious stock that you can't afford to throw out), I suggest placing a few small drops (i.e 20-50 microlitres) of 70% ethanol (with a pipette) directly on the mold itself. this may also kill some larvae, but hopefully only in the near vicinity.
For yeast: Yeast is a bit trickier. The flies eat the live yeast, so we want to add some. However too much yeast (or a very fast growing strain) can overcrowd the surface of the food and make it difficult for larvae to burrow. If this is happening, transfer the adults to a fresh vial and do rapid transfers as described above for mold. For larvae in the affected vial, I recommend adding some sterile water (in the wash bottles labeled H2O) scoring the surface of the food with a sterile forcep, and then adding some sterile (autoclaved) paper towel to it. This usually helps a bit. Usually the adults that emerge from this vial are pretty loaded with live yeast so it may be worth doing the rapid transfers for them as well.
TIPS & TRICKS:
- For fly pushing, Use thin brushes, big brushes are more likely to trap flies. We should have plently of brushes so it is ok to use them, and then place in disinfectant (70% ethanol is sufficient).
- Make sure all flies are cleared from plate between different genotypes
- Check brush before adding any flies to vials or bottles
- For weak stocks, note stock and check more frequently, keeping up on water or yeast can do wonders!
- "Score" food (scratch surface of food) before adding flies If possible, keep at 24 degrees
- We also have some easy to make artificial media that helps with weak stocks. Ask Ian first.
- For Drosophila melanogaster adding some mushed banana (usually in freezer) can also help. Talk with Ian first.