Скачать или смотреть Mastering master mixes - calculations & concepts

In my first lab rotation (where you try out different labs before choosing) the postdoc I was working with used this term and I’d never heard it but then I learned that it’s actually really common. So what is it?

blog form: http://bit.ly/mastermixesmultichannels

spreadsheet examples: https://docs.google.com/spreadsheets/...

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, you can prepare a “master mix” of all the “same stuff” - so you 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 shorted.

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 think 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.

For example, when I was radiolabeling RNA the other day (more here: http://bit.ly/radiolabelings) 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.
                   
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