Best practices for working with molecular biology enzymes

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Best practices for working with enzymes

- keep them on ice and/or in the freezer right up until you need them - prepare everything else and then go fetch it
- - often labs will have enzyme coolers with little ones for transport
- - keeping them in the cooler is good for when you’re trying to find an enzyme and don’t want to have boxes out of the freezer for a long time while you’re looking and you don’t want to keep the freezer door open
- - Don’t hold the tubes from the bottom - you’ll heat them up

- There will often be a reaction buffer that will need to thaw out - this can usually thaw safely at RT (room temp) but remember to account for this time-wise

- Unless specified otherwise, add the enzyme last
- - this has multiple benefits including
- —- allows you to more accurately time reaction times
- —- makes sure the enzyme is in its happy place when you add it (if conditions aren’t ideal for it it could denature (unfold), etc.)
- —- prevents off-target/alternative activities that can take place if all the reagents aren’t present yet
- Note: sometimes a protocol will specify what order to add things in, so follow it

- Often you only need really tiny volumes of the enzyme for each reaction - it can be hard to accurately pipet them but mastermixes can help - make a mastermix solution with more samples-worth than you need of everything that’s “generic” (buffer & other reagents), add enzyme last to that, and then add 1 reactions-worth of this mastermix to each sample
- - this lets you pipet larger volumes, have better consistency between the volume of each component added to each sample, and saves you a lot of tedious pipetting!
- - I typically make at least 1.5 extra reactions-worth of master mix. This way, if there’s a problem when setting up one reaction I have a backup (and a little excess to account for losses during pipetting and things getting stuck to tips and tubes)
- - note that there are certain instances where a protocol says not to make a mastermix - otherwise, it’s typically a good idea
- Alternatively/additionally, sometimes they come with an enzyme dilution buffer you can pre-dilute the enzyme in so you can then pipet larger volumes

- - different brands might have different concentrations and/or unit definitions so be sure to check if you’re going off a protocol that you’re using the right amount

- The enzyme stocks are typically in a high concentration (e.g 50%) of glycerol. This keeps them from freezing, protecting their activity. But it also makes them really viscous (goopy) so pipet very slowly, keep your pipet tip just below the surface so you don’t get goop on the outside of the tip, and watch the enzyme go into and out of the tip, making sure there aren’t bubbles going up and making sure that it all gets out. I typically like to mix up and down a couple of times with the pipet when I pipet it in, to try to wash out the tip. Since that tip is small though, I then pipet up and down with a larger pipet and/or finger flick to mix. DO NOT VORTEX (unless protocol says to) - that won’t make the enzyme happy…

- Remember to return the enzyme to the freezer ASAP
- If you treat them well, they’ll be good waaaay past their expiration date (but it might not be worth it to risk it depending on your sample… if possible do a positive control experiment first, maybe even a head-to-head old vs new)

- When you get an enzyme in the mail
- - it’s often on dry ice or at least ice gel packs
- - give it a quick pulse spin to bring everything to the bottom of the tube to minimize enzyme loss from wall-sticking and evaporation (minimize the surface area in contact with tube and air)
- Look to see where it should be stored (-80? -20?)
- Put the date you got the tube on the tube when you get it - sometimes they only put lot numbers and/or dates for a kit on the box not the tubes, so write it on the tube for later reference

- Know why you’re adding the enzyme - what does it do???? It’s interesting to me know and will help you if you need to troubleshoot. Also look up why it needs the various cofactors and things that go into your mastermix.