Overview
Solutions are a routine part of lab life. They are used every day in the lab for a variety of applications, from the stock solutions that are used in almost everyone's experiments, to solutions created specifically for your experiments and maintained as a short-term stock, to solutions mixed as part of experiments for one-time use. They can also include master mixes of multiple reagents created to develop more even plating of reagents and control experimental conditions. There are many different ways that these solutions are referred to and for a beginner in the lab, it can be incredibly confusing to hear these terms thrown around. Don't be overwhelmed! The solutions are simple to make and you will quickly learn to make them. However, make sure you understand how to do it because messing up your dilutions is an easy way to waste expensive reagents and interfere with good experimental results. Here, we'll go over the common terminology around dilutions and we'll discuss how to mix standard solutions.
The 'X' Dilution
This refers to the dilutions such as 1x, 5x, 10x, 20x, 100x, and so on. You can, of course, use any number but these are the most common. This description is used for stock solutions that are routinely used in the lab and that are required in large quantities. Keeping a small bottle of a 20x or 100x solution allows the lab to easily generate large quantities of 1x solution as needed. You can also do this for yourself if there is a reagent you use routinely. You make a 20x stock and dilute it out whenever you run out of your 1x stock to expedite the process.
This description means that the concentrated stock solution is 5, 10, or 20 times stronger than what you need it to be. You need to dilute these solutions down to 1x which is the concentration that you actually need. Using extremely concentrated solutions can destroy your cells or tissues, so ensure you dilute correctly before using.
To dilute these solutions think about the concentration as parts of a whole. This means if a solution is 5x and you need to get it down to 1x, you need 4 parts water and 1 part of the stock. That way, it will become 5 times weaker and get you down to 1x. If a solution is 10x, use 9 parts water to 1 part stock. Essentially, if you are using a stock, use 1 part of the stock and then add in all the remaining parts as water until you reach the final number for the stock.
For example, let's say we have a 10x PBS stock and we want to make 1000mL of 1x PBS. To do this, we first need to divide 1000 mL into 10 parts, giving us 100 mL per part. Then, we will add 9 parts water and 1 part stock. This means we will add 900 mL water and 100 mL of the 10x stock to make 1x PBS.
Let's do another example. We have a 20x sodium borate stock and we want to make 100 mL of 1x sodium borate. We start by dividing 100 mL into 20 parts, giving us 5 mL per part. Then, we need 19 parts water and 1 part 20x stock. This means we will add 5mL of the 20x sodium borate and 95 mL of water to achieve our desired 1x concentration. You can see how this would be hugely helpful to a lab. Having just 100 mL of sodium borate 20x stock would allow us to generate 2 liters of sodium borate 1x solution as and when we need it.
The 1:X Solution
The ratio is another very common way of expressing necessary dilutions in labs. This takes two forms - one is with x being a small number - say 1:5 or 1:2 - which works similarly to the dilution discussed in the section above. It means that 1 part out of 5 must be the stock, or 1 part out of 2 must be the stock. That means you would add 1 part stock + 4 parts water or 1 part stock + 1 part water respectively. For example, if I have an MTT stock solution and I want to make 10mL of a 1:10 dilution in PBS, I will take 1 part MTT stock + 9 parts PBS. 10 mL divided into 10 parts is 1mL each, so I will add 1mL of my MTT stock + 9mL of PBS to create the final stock.
The other form of the ratio is when x is a very large number. This is typically used to express antibody dilutions, as antibodies are extremely expensive and extremely potent. To use them, you will typically use dilutions ranging anywhere from 1:50 (at the lowest end) to 1:10,000 (at the highest end). Common dilutions include 1:100, 1:1000, and 1:5000. To calculate these, the 1 is essentially negligible so we don't calculate 4999 parts to the 1 part, we just add 1 part into 5000.
Let's go over this more clearly. Let's say that I am using goat anti-rabbit secondary for an experiment and the recommended dilution is 1:5000. I need 10mL of the 1:5000 antibody solution for my experiment to put my cells in overnight because I have 10 wells that need to stain and each one takes 1mL of antibody solution. So, what does 1:5000 mean? It means that for every 5000 microliters (5mL) that I use, I should add 1 microliter of antibody. We are talking about a tiny tiny amount of antibody relative to the total fluid in the container here. If you find yourself adding a lot, think again and double-check your calculations. Based on the above, for 10mL of antibody solution, I should be able to add 10mL of PBS to a tube and then put 2 MICROliters of antibody in it. I stress this because you do not want to waste antibodies. You will hopefully never find yourself in the position of having accidentally added 20 microliters of antibody to a dilution. However, if you do, you may have just used the entire antibody bottle.
The % Dilution
This refers to dilutions such as 1%, 5%, etc. When a dilution is described this way, it's almost always meant as a weight-per-volume dilution, i.e. that you put in a certain weight of a reagent and then dissolve it in a certain volume of diluent. The percentage is always based on a 100 mL volume (which makes sense because it's a percent). For example, if I said we need to make 5% milk in PBS for a western blot, that means 5% weight-per-volume, i.e., 5g of dried milk powder in 100mL of diluent (PBS in this case). You can then scale that formula up or down for the total volume you want to make.
For example, if we wanted to prep 100 mL of milk, we would just measure out 5g of milk and then measure out 100mL of PBS and mix it together. If we wanted to make 50mL, it would be 2.5 g with 50mL of PBS. If we wanted to make 1000mL, it would be 50g in 1000mL of PBS.
Practice Examples
For practice, let's work through the following examples.
1x PBS
Let's say we want to make 1000 mL of 1x PBS solution (which is the usable PBS concentration) from a 5x stock in the lab. To do this, we first divide 1000 mL by 5, giving us 200 mL. Therefore, we need to add 200 mL of the 5x stock to 800 mL of water (usually distilled purified water) in order to create a 1x solution.
MTT Reagent
Let's say we want to make a 1:5 dilution of MTT stock solution in PBS, and we want a total of 50mL of volume. This means 1 part out of 5 should be the MTT stock solution and the rest should be PBS. Therefore, we'll divide 50 mL by 5, giving us 10mL. This means we add 10mL of the MTT stock solution to 40mL of PBS, which will give us a 1:5 dilution.
Antibody Dilution
Now let's say we want to make a 1:1000 dilution of rabbit Notch1 in BSA for an experiment. We want to create a total of 2mL of antibody solution for our plates. To do this, we will first measure out 2mL of BSA and place it into a tube. Next, we'll think about how many microliters 2mL is equivalent to, which is 2000 uL. Now, a 1:1000 dilution means 2 uL of antibody in the 2000 uL of BSA. Therefore, we'll add 2uL of antibody into our 2mL tube of BSA, mix well, and then distribute to our plates.
1% Solution
Finally, let's say we want to mix a 1% solution of milk in PBS for a western blot. We want to make a total of 100 mL so we can cover all our blots. For this, we'll measure out 1g of milk and place it into a 100 mL tube of PBS. We'll mix thoroughly and then be ready to use for all the blots!
As you can see above, the math is easy!! The more you do these calculations, the more automatic it will feel in your mind. Eventually, it will just become second nature. Also, labs usually use the same dilutions over and over again so after a while, you'll just memorize the numbers you need for any given dilution.
Resources
To help you out as you get started, here are some online calculators that can help you confirm your numbers.
For more help, check out the protocol section! We have many common lab solutions listed with reliable and easy-to-use recipes. Most standard scientific solutions will have recipes available online through a quick google search as well. Pay particular attention to the storage and longevity of solutions. For things you use often that can be stored, it's helpful to make a large stock once rather than making them over and over again.
