Stock up on stock solutions so you can spend your time on the fun stuff! When it comes to “working hard or hardly working?” I definitely work hard, but I don’t want to work harder than I have to when it comes to the “boring stuff” like making buffers (pH-stabilized salt-waters that have what I need to keep the proteins I’m purifying happy). So I turn to stock solutions, which are like the biochemist’s version of space food – ready made, just add water! 

I’m kinda like a biochemical bartender – just like bartenders keep different liquors on their shelves that they can combine to make different cocktails, I keep different liquids on my shelf that I combine to make different solutions – my protein “customers” are picky & they have different “tastes” so I want to be able to make “custom cocktails to order” BUT I also want to save time by “stocking up” on the cocktails that get ordered a lot – without crowding up my shelf any more… If you thought my bench shelf was crowded with bottles, imagine if I kept everything at their working concentration (the concentration I want to use them at). Instead of stuffing my shelf with more bottles (well, stuffing it *more*) I stuff more molecules into each bottle, keeping high-concentration “stock solutions” (e.g. at 10X working concentration) that I can dilute (and mix and match) to order.  

It’s nice for a bartender to have the individual components separate so they can mix n’ match. But what if there’s high demand for some particular cocktails? It makes more sense to keep that cocktail pre-mixed to save time. You know you’ll need a lot of it. So you don’t just want to have 1 order’s-worth or you’ll still have to make it “from scratch” a lot. But you don’t want it to take up tons of space on your bar shelf because people still do order other stuff, just not as frequently. Instead of keeping tons of bottles of it, or one giant bottle, you can keep a more concentrated version of it that you just have to add water to – kinda like cans of condensed soup.

I don’t drink “alcohol” (yes water’s technically an alcohol since it has an “alcohol” group “-OH” but what we typically consider “alcoholic drinks” contain ethanol – more on that here…..http://bit.ly/2QWKWXp ) so I really don’t know what goes into those “real” cocktails, but that’s okay because my proteins don’t know either. And they don’t “ask for” scotch or gin, instead salts and pH-stabilizers called buffering agents are what typically goes in.

Let’s quickly get on the same page terminology-wise… (and remember my blog now has a glossary! – in progress…) http://bit.ly/bumblingbiochemistglossary 

A solution is just when you take (at least) 1 thing (the solute)(like a salt) and stick it in another thing (the solvent)(like water) and the solute molecules all give up their solute-solute interactions, replacing them with solute-solvent interactions, acquiring a full coat of the solvent. How much of the dissolved thing (solute) there is compared to how much solution there is is called the concentration and there are different ways it can be reported, like molarity which tells you how many solute molecules there are in a certain volume. More specifically, molarity (which is abbreviated with a capital M) tells you how many moles (where a mole is 6.02 x 10^23) of something are in 1L. more here: http://bit.ly/2KOLaLl

Molarity is an “absolute” concentration, it tells you how much stuff there is compared to the volume. But we can also talk in terms of “relative concentrations” which tell you [how much stuff there is compared to the volume] compared to [how much stuff you want there to be compared to the volume].  This is how we end up with bottles labeled “10X,” “5X,” etc. The high-concentration version is the “stock solution” because it’s the one you keep in stock. Its concentration is the “stock concentration” and the final concentration you want is the “working concentration” because it’s the one you want to work with when you’re doing your actual experiment (not just the prep part). 

Now, one more term: “buffer.” I’m a protein biochemist, so I purify a lot of proteins and study how they work. Above I mentioned how I can make buffers for my proteins to live in. But “buffer” is a super broad term… It’s kinda like how “growth media”  can be used to refer to any type of “food” you grow cells on or in (e.g. LB or TB for bacteria and those weird pink liquids for culturing mammalian cells in dishes). “Buffer” can be used to refer to any pH-stabilized liquid concoction that you put molecules into – either just to keep them happy (such as in the case of the buffers I store my proteins in) – or to allow reactions to occur (such as the buffers used for PCR (a technique where you make lots of copies of DNA in test tubes; PCR buffers contain, among other necessary ingredients, the Mg²⁺ required by DNA Pol to do the copying). The “buffer” name refers to the fact that, despite all their differences, these liquids all have some molecule that acts as a pH buffering agent – examples are Tris, HEPES, and sodium phosphate. 

Such “pH buffering agents” act thermostat-like to keep the pH at a set point – if the pH drops too low, they’ll act as a base and sop up the “excess” protons. And if the pH gets too high, they’ll act as an acid and let some protons go to make the solution more acidic. More on buffers here: http://bit.ly/phbuffers 

Stock solutions are huge time-savers because, just like you don’t want have to go ferment some whatever you ferment to get gin every time a customer orders a cocktail with gin in it, you don’t want to have to dissolve solid Tris – and then adjust the pH to right where you want it – every time you want to make a solution that has Tris in it. And, just like different gin-containing cocktails have different amounts of gin in them, different solutions I want to make have different amounts of Tris in them. So I want to keep a stock solution that’s at a higher concentration than any of the solutions need it at – with room to add any extra ingredients. 

So, for example, I keep 1M stocks of Tris at various pHs.

Having a constant pH is really important for reactions, so they’re key components of all buffers – but they’re just one component! 

Other common components are salts like NaCl & KCl, so I keep high concentration stock solutions of those as well (I find 5M NaCl & 3M KCl to be convenient).

To make a stock solution of one of these I start by looking at the container (or on Google) and searching for the formula weight (F.W.) or molecular weight (M.W.) (aka molecular mass) which tells me how much 1 mol of the thing weighs. So, for example, if I look at the container of NaCl (sodium chloride aka “table salt”) it tells me the F.W. is 58.44 g/mol. And if I want 1L of 5 mol/L, I will need 5*58.44g = 292.2g.

This is one of the situations where you can use dimensional analysis chart things to help. http://bit.ly/dimensionalanalysising

(1L)*(58.44g)/L*5mol/L = 292.2 g

But, don’t go measuring out 1L of water and adding 292.2g of salt. Remember the salt’s gonna take up some space and if you had 1L to start with you’ll have more than 1L after you add the salt. So, instead, start with a lower volume of water (maybe like 700 mL) in a beaker with a magnetic stir bar on a stir plate. And gradually pour in the salt. Don’t try pouring water on the salt or your stir bar won’t budge. And try pouring the salt in portions and giving it some time to dissolve. When you’re making these high concentration solutions, it can take some time……. Once it’s dissolved, pour it (but not the stir bar!) into a graduated cylinder and add water to 1L (and remember to measure from the bottom of the meniscus (the little smile of water that forms thanks to capillary action (water sticking to cylinder’s walls and creeping up them).

You can do similarly for KCl, or sugars, or basically anything. But those are still stock solutions of the “ingredients” – aren’t we wanting to stock up on the pre-mixed cocktails as well? Yes – and you can! If you make stock solutions of individual components, like salts and buffering agents you can mix and match to make different final “working” solutions. And you can even mix n’ match to get more “non-working” solutions.

But sometimes you want the same working solution a lot. In this case you can mix stock solutions of the individual components to get a stock solution of that mixture. For example, the running buffers we use for running SDS-PAGE gels & agarose gels (techniques to separate proteins and DNA, respectively, by size). You can reuse the running buffers multiple times, but we still go through a lot of it because we run a lot of gels.

So we make 10X or 50X versions of these that we then dilute when we’re ready to use them. 10X means the stock is 10 times more concentrated than you want to use it at. So to figure out how much of it to add, you need to reverse the X which means you need to divide. So if you want to make 1L (1000mL) you divide that 1000mL by 10 -> you need 100mL of the stock solution. Just dilute that to 1L with water and voila – 1X!

What if you thought a stock was 10X and it was really 50X? You can fix it if you find out soon enough (but you can’t go the other way as I’ve “learned” the hard way a time or too…). So, if we added 100mL of 50X, how much total volume do we really need to have a 1X working concentration?

Why don’t we bring our old friend C1V1=C2V2 in to help. This equation says that the initial concentration times the initial volume equals the final concentration times the final volume. We can plug in relative concentrations like “10X” and “50X” just as we would absolute concentrations (Molarity-type things). So,

(50X)(100mL) = (1X)(V2)

now we solve for V2…

V2 = (50X)(100mL)/1X

V2 = 5000mL = 5L

So now you can just adjust the volume to 4L and hopefully save your experiment!

Most of the time, you know the V2 you want (desired final volume) and you can use C1V1=C2V2 for each individual ingredient in your cocktail, using the same V2 and solving for each ingredient’s V1 (the volume you have to add). Then you can add up all those V1s and subtract that sum from V2 to figure out how much water you’ll need to add if you’re doing experiments on the tiny scale where you’re not working in graduated cylinders & filling to the line. 

In addition to these instances where you always want the same working solution, this type of stock solution is great for making a stock “background” for when you have multiple solutions you might want to make that have things in common but also differences. Like a wash buffer and an elution buffer for affinity chromatography (a protein purification technique) – that have the same salt & buffer concentrations but the elution buffer also has an addition competitor molecule to push your protein off the column it’s stuck to (get it to elute). You can start from the same high-X stock to get those “generic” parts and then add the unique parts. Since it’s at a higher x there’s still extra room to add extra stuff whereas if you started at 1x there wouldn’t be enough! http://bit.ly/2Fs8taY

These stock solutions save space and time (you don’t have to make it as frequently) and they often also have another benefit – keeping higher concentration versions of solutions is good because some solutions tend to be more stable at higher concentrations, kinda like extending the expiration date on that bottle. It’s harder for bacteria to grow in them because instead of a solution mimicking body-like conditions, which are what most of the solutions I make do, you have an overload. And since stuff isn’t growing in them they can last longer. 

Another precaution you can take is to vacuum filter them. We have this kinda bowl-like plastic thing that screws onto the top of our bottles that holds in place a disposable filter and hooks up to a vacuum line to suck liquid from the bowly thing into the bottle. 

Some stock solutions should be autoclaved (stuck in a super hot super high pressure steam cleaner) to sterilize them. This is especially important for things that are going to go into growth media (like TB salts you add to TB media for some extra-rich bacteria food). 

Theoretically you could make a stock solution at “any X” but there are some limitations…

  • physically – there’s only so much solute you can stuff into a certain amount of liquid – each combo has a solubility limit.
  • practicality- do you really want a 7.91x stock? Multiples of 10 are most convenient. Even numbers help keep numbers whole but 5s are ok cuz 2 of them gives you that convenient 10. Since it’s easier to think in 10s, I often start by dividing by 10 in my head then adjusting that value by doing what you’d have to do to get the real x to equal 10 – so if you have a 20x solution, you need to multiply by 2 (add twice as much as you’d add if it were 10x) and if it were 5x you’d divide by 2 cuz you have to divide 10 by 2 to get to 5. So to make 100 ml working solution from a 10x stock you’d need 10ml of the stock. You’d need 5ml from a 20x and 20ml from a 5x. 
  • accurateness – each time you make a measurement, you’re introducing the potential for measurement error. If every time you went to make a solution you had to weigh out each of its components you’d be introducing more inaccuracy. if you start from a stock solution then you just have to make a couple measurements each time. But didn’t you have to weigh those components out when you were making the stocks? Yes – BUT then you were measuring much higher amounts.  And, like an extra drop of water in a swimming pool isn’t that big of a deal but an extra drop of water in a teaspoon is much more noticeable, the measurement errors when you’re weighing out large amounts are less significant. Try as hard as you might, there’s always gonna be some error – even if you do your scientist duties perfectly, the measuring equipment itself has limitations. And if the measurement’s small even a “small” limitation’s “large” (think +/-1mL when you’re measuring 1L vs 5mL…). Imagine if you had to weigh out 1/2 a mg or pipet 0.1ul which might be all of that component that’s in the amount of solution you’re making…

Final notes…

Make sure that you check what the proper storage conditions are. Does it need to be fridged? Frozen? Shielded from light? (if you need to shield from light, you can wrap it in foil)

Especially for frozen stock solutions like antibiotics, it’s good to make aliquots if you make a lot more of the stock than you need to use each time. If you make aliquots (little single-use portions like 1mL portions in ependorf tubes) you reduce the number of times you have to freeze-thaw them. And if something happens to 1 of them like something starts growing in it or you accidentally pipet something into it instead of out of it or use a dirty tip or something you haven’t ruined it all.

My colleague makes fun of me because I give most of my buffers 3-letter abbreviations (like IRB for Insect cell Resuspension Buffer). HPB is my shorthand for His-tag purification buffer. And in the pics I show you how I make it (stock & working).

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0

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