Team:Hamilton McMaster/blog/2015-08-24
Preparing the Lab (For Science!)
Hello and welcome to A Fledgling’s Guide to Cell Bio! For returning birdies, welcome back! And for newcomers, don’t be afraid, we’re gentle creatures. My name’s Max and I’ll be your guide for today! So hop/read on as we begin our journey into synthetic biology lab work. First stop: Micropipetting!
1. How to Use a Micropipette: 1-1-2
I remember, when I first started lab work, how I knew nothing about micropipettes. What are these sleek-looking machines that scientists are always photographed using? How simple is it to use? And why does everything smell?
Well, if you’ve ever wondered, now’s the time to learn (we’ll get to that third question soon enough too)!
So this here is a micropipette, the ones we use at mGEM:
1. Make sure you have the right pipette and volume setting
Micropipettes have a certain range of volumes they work in. For example, the one in the image above can only work from 20 μL to 200 μL. Although the pipette is said to work to the extremes of its range, it’s safer to use a different pipette and work in smaller volumes, so pipette 1 uL twice from a 0.5-5 μL pipette instead of doing 2 uL from a 2-20 uL pipette. This avoids using the limits of the pipette, as these limits may be less effective and thus less precise at these extremes. The company may have already accounted for these limits, but it’s better to be safe than sorry! Once you definitely have the correct micropipette, simply rotate the Plunger (where you push with your thumb to release materials) until you arrive at the desired volume. If the numbers start changing in the wrong way (going up when you want going down), just start rotating in the opposite direction. If you feel resistance, stop rotating (or you might damage the pipette)! You might have unbeknownst reached the end of the micropipette’s range and you’ll need an alternative.
2. Attach the correct pipette tip
Different micropipette sizes will use different tip sizes. To add the tip, open the ip box, and, without touching the tips with your hand (they’ve been sterilized in an autoclave), gently yet firmly push the micropipette into the tip. When you lift the pipette, the tip should stay on it. If it’s your first time with these tips, make sure have the right size of tip (not too small). Test the tip size by trying to eject the tip by pushing on the rear button (known as the Tip Ejector). If the tip shoots off (be careful!), great - you have the correct tip! If the tip stays on, then you have too small a tip size. If the tip size is too big, it wouldn’t fit on in the first place.
3. In air, push ‘once’ (1)
While in the air or fumehood, push down once on the plunger and hold it there. This will eject the air from the pipette and prepare you for the next step. It’s important to move quickly from one step to the next, as the longer you leave the sterilized pipette tip in the air, the more contaminated it becomes.
4. Insert into material and release ‘once’ (1)
Insert the tip into the material you want to suck up, and then release your hold on the Plunger. This will cause the material to rise up and into the micropipette.
5. Eject material into container by pushing ‘twice’ (2)
Move the pipette tip into the container you want to add the sucked-up material into, and now push the Plunger completely down. There are two stages to a micropipette’s material ejecting ability. During Step 3, When you only push down once, you expunge the amount of air you set on the micropipette. This means that, in Step 4, you suck up the same amount of the material. Now, in Step 5, by pushing ‘twice’, you push a little bit more air than the set volume. This ensures that all the sucked-up material is expunged from the micropipette tip. This may not be super important for some non-viscous liquids, but very precise small volumes of liquid, as well as viscous fluids like glycerol, will benefit greatly from this two-step push.
6. Without releasing, remove the micropipette from the container.
Do not release the Plunger or you’ll just suck everything back up again! Simply remove the micropipette from the container, and then eject the tip, by pushing the Tip Ejector on the back of the micropipette, into your prepared Waste Tip Container.
7. Repeat as necessary
This concludes the basic use of a micropipette! Just repeat these steps (1 push, 1 release, 2 push eject: 1-1-2) each time you use a micropipette. Just remember: unless it’s the exact same material and material source, and you’re adding it to a sterile and empty container, always change pipette tip between uses! Otherwise you risk contamination of source material and your experiment.
2. DNA Resuspension: The First Step to Re-animation Sciences
Now, quick re-cap to make sure we’re all on the same page! DNA is short-hand for deoxyribonucleic acid. You can learn more here, but for now you just need to know that it was discovered not too long ago (20th century) and is made up of two sugar-phosphate backbone that run antiparallel to each other. The nucleotides, Adenine, Thymine, Cytosine, and Guanine (A, T, C, and G), connect in pairs (A-T, C-G) to each other using hydrogen bonds (which is why DNA is so relatively stable compared with other hugely complex molecules!).
So now that we know DNA, what’s resuspension? Well, image you’re sending someone some foods. Like apples. But they’re far away. Like International Space Station far away. You can’t just send the astronauts fresh apples - they’ll rot during the long journey! So instead, we have things like freeze-dried apples. By removing the water content from biological materials, the stability of the materials increases.
It’s just the same with DNA. For us here at mGEM, our first DNA samples came in these here fancy plates:
The DNA samples in each well are dry (via miniprepping - more on that in the future) and dyed (cresol red), and are stable at room temperature. We add nuclease-free water to re-hydrate the DNA, allowing for it to be manipulated and pipetted.
There are 384 wells per plate, and the aluminium foil covering protects the DNA from contamination (we’ll revisit this later). iGEM distributed not one, not two, but FIVE plates in total to each team! Super cool! And that brings us to our second stop:
3. Proper storage of DNA, enzymes, buffers, antibiotics, and competent cells: Super cool!
Keep the DNA stored at -20°C. Otherwise the DNA might start degrading itself. DNA, enzymes, and buffers should be stored in a -20°C freezer (unless specified otherwise by the supplier). Some antibiotics should be stored in 4°C, although some can be stored at room temperature (e.g. Kanamycin). The dried-state of an antibiotic might or might not be able to be stored at room temperature. It’s important to read the SDS (Safety Data Sheet) and information provided by your supplier before handling, storing, and using any materials! These temperatures are just general ideas based on what we here at mGEM use.
When you want to use your DNA and enzymes, they’ll need to be thawed, but you still want to keep them cool. So prepare a styrofoam (or any other insulation) box filled with ice. Place the DNA and enzymes into the ice, where they’ll thaw in a few minutes. Buffers can be thawed in water. Although thawing can be accomplished by using body temperature (holding the tubes), it’s not ideal because it’s very fast and very warm and this could shock your materials. So use ice or water, as appropriate, when possible. Remember: rushing and panic are two major factors that can ruin an experiment!
Now, the storage of comp cells is a slightly different story.
Wait, comp cells?
Yes, comp (or competent) cells. These are the E. coli bacterial cells in which you’ll be inserting your plasmids. You might not necessarily be using E. coli, as alternatives such as yeast or mammalian cells exist, but this is what we use for mGEM 2015.
Comp cells should be stored in a -80°C freezer. You’ll easily notice these freezer by how awesome they look:
DH5α type cells: These are a special and commonly used variant of E. coli cells that have had their cleavage genes knocked out, allowing them to accept any plasmid. This is key during the first portion of experiments, when trying to clone and manipulate plasmids - you don’t want your cells to reject or change your plasmids!
BL21 type cells: These are another special and commonly used variant of E. coli cells that have had their protease enzymes knocked out, allowing unnatural proteins to be kept intact. This is good for protein production, and is key for latter portions in experiments when analyzing the success of the plasmids.
4. Preparing Agar Plates and Overnight Cultures:
We use Lysogeny Broth, commonly known as LB (and technically not several other common names), as our nutrient-containing growth medium for bacteria (a.k.a. this is the world in which our bacteria obtain their nutrients from). It’s a barely-clumpy powder in solid form, and slightly orange-brown in wet form. It is extremely smelly, and though most of us dislike the smell, some people do quite enjoy it. Still, I don’t recommend you sniffing the LB.
Depending on your manufacturer of LB, the LB composition and thus recipe to make plates and tube cultures may vary, although the recipe should be on your LB container.
LB agar plates, as shown below, are solid versions of LB mixed with agar. The LB provides the nutrients, while the agar acts as the solidification agent. These are made in common but disposable petri dishes. The bacteria can be spread and streaked and picked from these plates (more on this in later posts!). We use a black permanent marker to detail important information on each plate: The medium (LB Agar), the antibiotic (Chloramphenicol), the date (not shown), and the plasmid under research.
LB in its liquid state is also used for overnight cultures. These are cultures in which a single bacterial colony (looks like a speck on a plate) from a streaked LB agar plate is added to create a huge number of bacterial clones. These cultures are left to incubate at 37°C in a shaker (to aerate the bacteria) ‘overnight’ (or about 24h). Below is an image showing an LB overnight culture tube that is full of bacteria, and is hence cloudy (the above portion is clear as it has no bacteria). Optical density is calculated based-off of this cloudiness. Because we re-use these tubes (after sterilization via auto-clave), we use green tape to label the tubes. Because we only use tubes for LB overnight cultures, we only label with the date and the plasmid (we know the antibiotic). However, because we don’t label it with everything, it is now more than ever extremely important to keep an up-to-date lab notebook (we have a snazzy black one with a nice iGEM sticker on the cover)!
It is important to note that these LB agar plates and LB overnight cultures are not only LB (and agar if appropriate), but also include an antibiotic, known as your Selection Marker. Only bacteria with plasmids containing the proper antibiotic resistance gene will survive in these mediums. This selectivity allows you to carefully select only those bacteria, or specimens, that have your plasmid (which contains this resistance gene). However, caution must be taken, as each antibiotic and its respective bacterial antibiotic-resistance mechanism differs. For example, resistance against Ampicillin (Amp) causes the destruction of Amp, and therefore cultures using Amp can only be incubated for 24h. Any longer and you risk other unknown bacteria growing in the medium, and thereby ruining the experiment sample.
Summary
Wooaah that was a long post! Kudos if you read it all! If you skimmed over it, no problem, here’s a summary table to compile all the most important bits:
-
Micropipette
Remember the 1-1-2 1 push out air 1 release to suck up material 2 push to eject everything -
DNA Resuspension
Add nuclease-free water. -
Storage
Antibiotics 4°C DNA -20°C Enzymes -20°C Buffers -20°C Comp cells -80°C -
Preparing Plates and Tubes
Use the LB recipe found on the specific container. Use LB to make LB agar plates and cultures.