If you want to master lab work, master mixes are your friend. And stock solutions can be the solution to weighing and dissolving that seems to never end! I might look super energy-filled in my pics, but today I’m actually super exhausted (but exhilarated by some exciting experiments) – and can’t imagine how much more tired I’d be if I had to make all my molecular concoctions from scratch every time. Instead, I premake “stock solutions” and then, when I want to make up a reaction mixture, I mix them together. And to make things even faster, if I’m making a mixture where only one component changes from “recipe to recipe,” I mix together the “similar parts” to make master mixes – kinda like making cookie dough and then adding chocolate chips to some and almonds to some.
We’ve been talking a lot about theory, but what about the day-to-day? I spend a lot of a lot of my days transferring liquids from bottle to tube, to tube, etc. But what I really care about is what’s going on in the tubes after I mix a bunch of stuff together. So I want it to be as fast as possible to get to the mixed state where I can try to figure out how the molecules played with each other. A couple of major time savers in this regard are “master mixes” and “stock solutions” and while it might sound kinda boring, it lets you get to the exciting stuff faster! So it’s a practice you want to master.
Stock solutions are like the biochemist’s version of space food – ready made, just add water! But how much to add? X marks the concentration factor on my stock solutions & clears up more spots on my shelf, which is is a bit like a biochemical “bar” – 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 concentrations!
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, commonly buffers, which are like pH-stabilized salt-waters that I can bathe my proteins in to keep them happy. More on them here: http://bit.ly/2X4Yhz7
It’s nice to have the individual components separate so you 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.
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.
A solution is just when you take 1 thing (the solute)(like 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 here: http://bit.ly/2OwH4LJ
Stock solutions are just where you make a solution of something at a higher concentration than you’ll actually want to use it at (more solute molecules stuffed into that volume) – and then you add however much of it you want to get to your desired final concentration – we call the desired final concentration the “Working concentration” because it’s the one you actually want to be working with when you’re doing the experiment work not just the prep
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 NaCl (table salt) every time you want to make a solution that has NaCl 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 NaCl 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.
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. 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!
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. 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
In addition to saving space and time (you don’t have to make it as much) 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 mimic body like conditions, which are what most of the solutions I make do, you have an overload. Since stuff isn’t growing in them they can last longer. Another precaution you can take is to vacuum filter them.
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.
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…
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.
Make sure that you check what the proper storage conditions are. Does it need to be fridged? Frozen? Shielded from light?
Especially for frozen stock solutions, 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 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)
Another solution-making solution for some situations is the “master mix.” Often the lab you have to set up a bunch of reactions that are almost the same – they just differ in 1 or 2 things – like you want to radiolabel different RNAs and all the things you need for the labeling (water, buffer, kinase, ATP) are the same, but the RNAs are different. Since all the reactions are the same except for that thing I can prepare a “master mix” of all the “same stuff” – so that I only have to add 1 thing per different thing instead of 4
It’s kinda like if a company has a form they need all their employees to sign. If the company had to type up the form individually for each person, that would take a really long time – and each time introduces the chance for the company to make a typo – and it can be pretty easy to get exhausted and or boredly mind-wandery, etc. after typing the same thing out over and over. The lab version of this is what I call “pipetting apathy”
So, the company instead makes 1 form and just leaves room for the employee to sign. If there are a couple of things that can vary (e.g. maybe they need to print their name and sign, and/or write down the date) they can leave space for those too.Then they only have to type it up once, can distribute this single document to all their employees, and don’t have to worry that they accidentally gave 1 employee a form where they left out the “not” before “liable” – getting their employee to sign that the company *is* liable for damages… The company doesn’t just print out the number of copies they need – they print extra so that if someone messes up their form (pipets the wrong thing or wrong volume, etc.) or loses the form (drops a tube or something) they don’t have to type up a whole new form.
When it comes to pipetting the situation’s a bit more complicated too because instead of having defined sheets, everything is mixed together, so it’s more like ladling punch. Each time you ladle, a bit gets stuck on the ladle, some may spill, some evaporates, etc. so you want to make a bit more than you need (e.g. if you have 20 guests & you want each guest to get exactly 1 cup of punch, you don’t want to make exactly 20 cups-worth of punch or the last person will get jipped.
So when making master mixes, you always want to prepare for more reactions than you actually need – because every time you pipet, some gets stuck to the pipet tip, some can evaporate, etc. & you want to make sure you have enough. If all the stuff’s cheap, it’s good to make enough for a little more than 1 extra in case you mess up on 1 reaction you have enough to redo it. But when the reagents are expensive and/or radioactive, I just make enough extra to account for some minor losses.
If it’s something you do a lot and/or there are lots of components with decimals and stuff, you can even make a spreadsheet where you have a “per reaction” column and then a “total column” with the amounts you get if you multiply it by a “conversion factor” you can change (if you’re using excel, use F4 after you click on the well you want it to multiply by (the conversion factor so if you try to copy the formula it still knows where to look).
Another thing to watch out for – remember that the total reaction volume is the master mix PLUS the unique thing. So for a 50uL reaction volume, remember that you don’t add 50uL of it to your thing.
So, when I was radiolabeling RNA the other day (more here: http://bit.ly/2lb0O9U I I premixed the buffer, water, hot ATP, & PNK – and added 48ul of that directly to 2uL of RNA to get to the final reaction volume of 50uL. I do this type of “master mix” thing a lot. It’s a big thumb-saver & tip-saver and it helps ensure that all the tubes are getting the same amount of everything and you don’t accidentally skip one of the components for one tube, etc.
sorry for formatting and repeating a few things – speaking of using remade parts – I smashed together stuff from a couple past posts for today because it was a long day in the lab (which included using stock solutions and master mixes)