Lab Setup
How to set up a classroom research laboratory:
When starting my course, I had a budget to purchase equipment and supplies that was very generous for a high school course, and that allowed me to purchase good quality, sturdy equipment in some areas, but that required me to be frugal in other respects, compared to what is done in university labs. I chose to prioritize quality when the quality would impact either the ability to fit experiences for multiple student groups into a class period (Nanodrop; 96-well thermal cycler) or when the durability in student hands might matter in the long run (agarose gel boxes). My classes are up to 18 students where the labs are held in the classroom and where the classroom is shared with other courses (7th grade - 12th grade). Basically, my classes are able to do just about any bacterial or yeast work or genetic engineering. In those regards, I can do almost anything in the classroom that I could do in a university lab. The main exceptions are that I'm not set up to dissect yeast tetrads or to do Southern blotting. We also do one C. elegans RNAi lab. I have taken advantage of a friend's kindness in allowing me to store strains in her -80 freezer at Rockefeller, which gives me an excuse to visit her once a year. I've also been lucky that Jef Boeke, a former mentor from Johns Hopkins, now at NYU, has given my students the opportunity to collaborate in a small way on Sc2.0, the synthetic yeast genome project. Jef also kindly allowed me a bench in his lab one summer, which in addition to generally being helpful, allowed me access to a yeast tetrad dissection scope and a CHEF gel apparatus for running out whole yeast chromosomes.Here are my notes on how I set up the lab for the students:
Student equipment kits
Per pair of students:
- In the basket (each pair's materials marked with a unique color of lab tape):
- 2 p-20 pipets
- 2 p-200 pipets
- 2 p-1000 piptets
- 1 box of yellow p-20/p-200 tips, sterile
- 1 box of p-1000 tips, sterile
- 1 small plastic jar of sterile microcentrifuge tubes (Straight-Sided Polypropylene Jars with Screw Caps, 16 oz/500ml)
- 1 microcentrifuge tube rack
- 1 block rack with 50ml, 15ml, 1.5ml, and 0.2ml tube openings
- 1 foam flotation rack
- 1 small plastic box for microcentrifuge tubes (room temperature)
- 1 small plastic beaker for waste (250mL)
- 1 fine Sharpie
- 1 extra-fine Sharpie
- 1 small roll of a unique color of lab tape for identifying these supplies
- 1 small (7x8cm) Owl EasyCast B1A mini gel box
- 1 small plastic box for microcentrifuge tubes (stored at -20°C)
- 1 manual universal pipet filler
- 1 test tube rack
- Sterile toothpicks, wood sticks, glass beads (3mm) - I store these in glass test tubes and make aluminum foil "caps"
- For PCR
- 1 ice bucket - I use the two halves of the small NEB styrofoam shipping containers as two student ice buckets.
- 1 MicroAmp® 96-Well Tray for 0.2ml PCR tubes that fits the Applied Biosystems® 2720 Thermal Cycler - I'll combine all student tubes into one tray to put in the thermal cycler, but each group needs their own tray that can sit in their own ice for reaction setup. Having one base for the teacher is good for freezer storage, but students don't need them since their tube racks will be sitting on ice.
- For DNA gel extraction
- Hand-held blue LED light
- Small glass plate to place under the gel when cutting
- Uvex SCT-orange glasses to block the blue light - these aren't quite as good as the ones from Clare Chemical, but are more than sufficient.
- For the C. elegans RNAi induction experiment
- 1 dissecting microscope
- 1 worm pick made from platinum iridium wire, 0.25mm=30Gauge (90%:10%), and a glass Pasteur pipet
- For the yeast cdc13-1
- 1 regular microscope (shared from other classes)
- 1 hand tally counter
- 1 Hemacytometer for counting yeast (we already had a class set)
- 1 counter pen for counting colonies on plates
Per group of 4 students (2 pairs):
- 1 microcentrifuge (Eppendorf minispin)
- 1 vortex
- 1 power supply
- 1 protein gel box that can accommodate 2 gels: BioRad Mini-PROTEAN Tetra Cell (2-gel)
Equipment for the class:
- Teacher supplies:
- Tuttnauer Manual Autoclave 2540M
- p-20 pipet
- p-200 pipet
- p-1000 piptet
- Drummond Portable PipetAid
- hot plate with stirring function
- balance
- weigh boats
- weigh paper
- stir bars
- beakers and graduated cylinders for media prep
- autoclavable 125ml, 500ml, and 1000ml bottles
- aluminum foil
- autoclave tape
- lab tape
- Hot mitts
- syringes
- syringe filters (2µm)
- filter sterilization units (2µm)
- faucet vacuum attachment for filter sterilization. This is a very low cost alternative to setting up an actual pump that's great if you only need to use it occasionally. Carolina item # 711974.
- pH meter - I use the Pasco probes we have for Chemistry class
- cryogenic vials (for strain storage in your friend's -80°C freezer). I've also had some success storing bacterial strains in 25% glycerol at -20°C in 1.5ml microcentrifuge tubes in the labtop coolers for a couple years, but this isn't a very long-term option.
- 12-channel p-20 pipet (obtained later with a student award)
- 12-channel p-200 pipet (obtained later with a student award)
- -20°C manual defrost freezer (Automatic defrost cycles create temperature changes that are harmful to enzymes, so be sure to purchase manual defrost). This should be fairly large.
- Two Nalgene-20°C Labtop Coolers, Holds 32 tubes. I store enzymes in these to maximize their shelf life. These minimize enzyme temperature fluctuations. They also serve as a backup for short-term power failures since they're supposed to stay cold for 24 hours.
- 4°C refrigerator (I've made a dorm-size fridge work for storage of chemicals and microbe strains; a larger fridge would be needed if you intend to store lots of plates).
- Ohaus analytical balance (0.0001g) (shared with other classes)
- Clare Chemical Dark Reader Blue Light Transilluminator DR46B. Having this is fantastic, not only for DNA gel imaging (avoiding mutagenic UV light), but also for the class to use to visualize fluorescent proteins on bacterial plates or in tubes. A side note is that this uses an AC/DC adapter to convert to direct current, so the blue light doesn't flicker at all. This is good for photography and also means that it won't produce the LED flicker to which some people (like me) are sensitive in terms of headaches.
- Gel-imaging box with digital SLR camera and Dark Reader orange lens camera filter. I didn't want to spend my money on a fancy gel illumination system - I preferred to save it for the Nanodrop. You could go really inexpensive by just laying a smart phone on the orange filter that comes with the Dark Reader and holding it over the Dark Reader to take photos in a darkened classroom. I've done this when I've been too lazy to set up the camera. The quality is OK, but isn't great because images can be fuzzy and you can't easily control exposure. I wanted to make it approximate lab quality without the lab price. I purchased a refurbished Canon Rebel digital SLR camera from B&H that could be connected to a laptop and used to take photos by triggering the shutter and controlling exposure through desktop software. In 2011, this was only possible with SLR cameras, but I don't know it there are better technologies today with less expensive cameras. A camera mounting bracket (also from B&H) allowed me to attach the camera to a pole. They helped me in-store to pick out something appropriate. As you can see in the picture, this one has a sliding bracket mount (right of bracket in picture) that's helpful for positioning the camera properly in the hole. I also purchased a corded adaptor that allowed the camera to be powered from the wall outlet so I didn't need to worry about batteries. Initially, I rigged all of this up using a cardboard box with a hole cut in the top for the camera, with the Dark Reader sitting inside. This was helpful for figuring out dimensions, and then a colleague and students built us a box out of black plexiglass (~$100 for materials). This has worked out really well. I have the camera software automatically save the photos to a shared google drive folder and just tell the students their photo number when we snap the picture. I use the same camera setup (just using a ring stand pole without the plexiglass box) to photograph protein gels and bacteria/yeast on plates.
- 1 medium (9x11cm) Owl EasyCast B1 agarose gel box
- 1 extra large (23x14cm) Owl D3-14 Wide agarose gel box (obtained later through a student grant). This is extremely useful if you start doing any larger-scale original research. It accommodates up to 200 samples and can be loaded with a multichannel pipet.
- White light box for protein gel imaging
- Eppendorf 5702 Centrifuge or another clinical centrifuge for pelleting microbe cultures. This centrifuge is generally useful and for this class, I started to routinely have students prep 5ml of bacterial culture - pelleted in this centrifuge - when using Qiagen minipreps because many of the plasmids we work with (such as fluorescent protein gene plasmids) aren't particularly high copy number and this helps to maximize yield. This kind of centrifuge is also necessary if you intend to prepare your own competent bacteria, although a cooling version would be even better.
- Applied Biosystems® 2720 Thermal Cycler
- Nanodrop 2000 spectrophotometer. This spec is expensive (I got a deal on a demo model), but allows you to quantify DNA using only 1µl of sample. You just place your 1µl on the pedestal, measure, and use a Kimwipe to wipe off the pedestal between samples. Because samples don't need to be diluted in cuvettes and because it's rapid to process each sample, having this spec allows students to spec their own samples during a class. This spec was my biggest splurge item in my budget, but I'm very pleased that I went for it. It works well for students. Also, being able to easily quantify even very low DNA concentrations helps you to have a functional genetic engineering lab, since you often need to control the molarity of DNA that you're combining when engineering. If you don't intend to do genetic engineering, you wouldn't need a spectrophotometer. Alternatively, you could quantify larger molarities of DNA on agarose gels by comparing them to a ladder with known DNA concentrations.
- Ice machine (fine pellets)
- Microwave
- Waterbath
- 2 heat blocks for microcentrifuge tubes
- 1 large incubator that could hold a magnetic shaking platform
- Small incubator with Rotational mixer (LabNet Mini Incubator With LabRoller Rotator) - Note that having 2 incubators was often helpful. However, the little Rotator spins slowly and does not create enough aeration to allow its use for culturing bacteria. I found this out the hard way and then purchased the Orbi Shaker Jr. to fit into the large incubator we already owned. The LabRoller Rotator is useful for mixing solutions, but not for microbe culture.
- Cooling incubator - recommended, but not required for C. elegans work. I've tried two different lower-cost versions of dual heating/cooling incubators, but two of each of them have broken and stopped cooling. For one of them, this may have been due to long-term high-heat use by a student in another class. Now, I make sure that we only use this for cooling C. elegans work a few weeks each year to try to preserve it.
- High-speed orbital shaker (Benchmark Scientific Orbi Shaker Jr with Magic clamp platform) that fit inside the large incubator we already owned. I added a metal platform for it to sit on and zip tied it to the metal rack in the incubator to keep it from "walking" out. It just barely fits when turned 90 degrees. If I were purchasing everything new, I would consider an incubator with the shaker built in. I had initially tried to get by with the LabRoller Rotator for bacterial cultures, but their growth wasn't good enough. I had to upgrade, and I'm glad I did because this shaker is so much better generally, and also accommodates larger cultures using flask clamps.
- 2 of 15-ml magnetic 32 test tube racks
- 8 of 250-ml magnetic flask holders
- The platform has holes at each corner, so I was able to use mini bungee cords to secure microcentrifuge tube racks when needed.
- Stovall Belly Button Orbital shaker - for gel/membrane staining
- 20-L polypropylene carboy for a class stock of agarose gel running buffer
- Hand-held long-wave UV light (for visualizing BFP)
- UV Stratalinker - I purchased an inexpensive one on eBay and we use it to induce DNA damage.
- Vernier SpectroVis Plus Visible spectrophotometer/fluorometer and cuvettes- we've used this as a fluorometer to quantify fluorescent protein emission in purified protein samples.
- Microscope camera - for the C. elegans RNAi induction lab
- Nalgene square 1-oz bottles - these are fantastic for distributing solutions to students because they're unlikely to spill. I use them to hold multiple student aliquots of Qiagen miniprep kit solutions. I label each solution bottle and cap with a unique color of sharpie to maximize the likelihood that the right cap will go back on the right bottle. I store the buffer P1 bottles in a ziploc bag (to keep them together and minimize evaporation) in the refrigerator.
- Glass Pasteur pipets (9") and bulbs (for teacher use when pH-ing solutions with concentrated acid and for creating C. elegans worm picks)
- Waste bin for material to be autoclaved (8 qt)
- Autoclave waste bags (14"X19")
- Glass spreaders - for harvesting bacteria in the Fluorescent Protein Analysis lab
- Plastic flat-bottom containers for protein gel staining - I use disposable plastic sandwich-size containers with lids (from the grocery), which allows me to stack multiple containers on top of a fairly small platform shaker.
- Plastic floating microcentrifuge tube bubble racks
- Tube locks for microcentrifuge tubes for use when boiling samples
- Freezer boxes for microcentrifuge tubes
- Nalgene wide-mouth storage bottles and steel balls for shaking and storing yeast amino acid dropout mixes
- Other disposables:
- Extra sterile tips
- Extra jars of microcentrifuge tubes
- 1.5 ml microcentrifuge tubes
- Screw cap 1.5 ml microcentrifuge tubes (for boiling in the ninhydrin test in the Fluorescent Protein Analysis lab)
- 0.2ml PCR tubes
- 8-tube strips of 0.2ml PCR tubes and strip caps (this kind of tube is required for submitting sequencing samples to Genewiz)
- Razor blades
- Parafilm
- Kimwipes
- 100mm petri dishes for most work
- 60mm petri dishes for C. elegans growth
- 2ml disposable pipets
- 10ml disposable pipets
- 25ml disposable pipets
- 15ml polypropylene centrifuge tubes
- 50ml polypropylene centrifuge tubes
- 17X100mm Sterile polypropylene culture tubes - I use these for all small-scale microbe liquid culture - the fairly large diameter helps to maximize aeration and the caps stay on with appropriate venting better than those of 15ml centrifuge tubes. I've tried smaller tubes, but the aeration isn't as good.
- Plastic pellet pestles (for GMO detection lab)
- Toothpicks
- Wood sticks
- 3mm glass beads
- Sterile Q-tips for spreading a lawn of bacteria in the Fluorescent Protein Analysis lab
- Sharpies
- Disposable nitrile gloves (for student/teacher use with agarose gels)
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| Homemade gel imaging system |
Water
We didn't have a reverse osmosis water tap and I decided setting up a water purifying system wasn't worth the cost for our needs. Instead, we purchase commercially-available distilled water that we use to prepare culture media and most solutions. Good prices with delivery are available through office supply stores. We purchased a liter of bioreagent-quality water that is DNase-, RNase-, and protease-free that we use for PCR, restriction digests, and other DNA work. After 8 years, we still have about half of the original bottle left.
Gel Electrophoresis
For gel electrophoresis, I routinely have students use sodium boric acid buffer (Brody and Kern, Biotechniques 2004) in teaching labs because it can tolerate a very high voltage which facilitates students having the time to load, run, and photograph gels within a single class period. We use Biotium's GelGreen for DNA staining due to its high sensitivity (more sensitive than ethidium bromide) and the significant danger of using the mutagen ethidium bromide, particularly with children in the classroom. I like GelGreen better than other green DNA stains from a safety perspective because it's supposed to have a large chemical group attached to it that prevents it from crossing cell membranes. However, I still make students wear gloves and treat it like it is dangerous, just to eliminate any possible risk for them or for other children using the classroom. It's stable enough at room temperature that I've had no problem using one tube over the course of multiple school years with no decrease in efficacy. I also like the additional classroom safety (and lack of DNA damage) of being able to use blue light to detect GelGreen (although it also works with a UV transilluminator). GelGreen is available from various suppliers including Carolina Biological (Item # 217305).
Autoclaving for the classroom laboratory
Disclaimer - you should follow all of the manufacturer's instructions and use at your own risk. |
| autoclave tape changes color |
Since it's manual (lower cost), for a dry cycle I need to remember to switch it to exhaust after the timer has gone off, but before the pressure comes all the way down so that the high pressure can force the water back out of the autoclave chamber when switched to exhaust. When I forget, I need to turn it back on (about 15 more minutes on "sterilize") to get the pressure back up so I can try again to remember. Then I'll turn on the heating element again to dry for 45 minutes with the door cracked open on the exhaust/dry setting. This gets things mostly dry, but I tend to leave containers sitting at room temperature to dry more for another day or so before using tips or securing caps. Remember that all caps need to be loose on dry cycles too so that the steam can sterilize the interiors and so that the pressure can equalize inside and outside of the containers to prevent implosions. That's why microcentrifuge tubes are always autoclaved with their caps open.
I like that the size of the chamber and trays are a good size to fit the tip boxes and tube jars that I use:
Typically for plates, I autoclave a half-filled bottle of 2X agar separately from a half-filled bottle of 2X media. I can store these indefinitely on the shelf. Then, when I need to pour plates, I just microwave the agar and there's plenty of space above the liquid to accommodate some bubbling (you still need to watch and stop the microwave each time the bubbling starts). When the agar is molten, I pour in the room temperature 2X media into the 2X agar, swirl, and the temperature of the resulting 1X media is good for pouring plates. Or, if I want to pour plates immediately, I'll take the 2X media bottles out of the autoclave, finger tighten their caps, and let them cool quickly in a pan of water, while leaving the 2X agar bottles sitting in the autoclave with the door closed to keep them hot. When the 2X media is about room temperature, I'll combine it with the hot 2X agar and pour plates.
I use solid-bottom metal pans for autoclaving bags of waste (important in case of much greater likelihood of spillage).
The one thing that is not ideal about this autoclave is that 1000-ml bottles are too tall to fit in the autoclave when sitting on the trays. I've made it work, though, because I figured out that I could sit these bottles in polypropylene baskets and fit 3 in the autoclave at one time (I rarely need to do this - I mostly use 500ml bottles instead). This isn't ideal - try at your own risk- and you should be very careful that they're sitting stably. If you'll need to autoclave bottles of this size on a regular basis, you should buy a larger autoclave.
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| 1-liter bottles |
Microwave
For safety, it's extremely important that your microwave is designated only for lab use and not for food use. Because you might be autoclaving agarose in containers that may have trace amounts of DNA stains, you can't also use the microwave for food. Any DNA stain, even a "safe" one, has some chance that it might act as a mutagen since its function is to bind to DNA. Food should never be exposed to DNA stains. If you use ethidium bromide in your lab, you know that it's definitely a dangerous mutagen.
Waste
I collect any microbial waste from the class and autoclave it before disposal. This 14"x19" size of autoclave waste bag fits in the above autoclave. The waste bucket for those bags is 8 qts. When autoclaving waste, I remove the tray holder from the autoclave so the bags won't snag on it. I put the waste bag, loosely secured with autoclave tape, in a solid-bottom metal pan to collect any spill when autoclaving. I think the metal pan I found for this is an old dissecting instrument tray.
Molecular Biology and Biotechnology Course Support
Most companies were willing to offer discounted prices when I asked and explained I was purchasing for a high school.
I made purchases through formal course support programs at the following companies:
New England Biolabs - free reagents after documenting their use in a course.
Gilson - free refurbished pipets (through a rep I met at a conference)
IDT - a limited number of free oligos (through my sales rep)
Promega - 50% off for courses
Stovall was kind enough to donate a used Belly Button orbital shaker.
I made purchases with standard education pricing through:
Fisher Scientific (Fisher Science Education) - discounts visible after login with an education account.
BioRad (protein gel supplies)
Others who provided discounted rates:
Life Technologies in 2011 for the thermal cycler (since acquired by Fisher)
Qiagen (with quote through my sales rep)
Sigma-Aldrich - antibiotics, canavanine (with quote through my sales rep)
Nanodrop (refurbished Nanodrop 2000 through my sales rep; now available through Fisher)