Pelleting tips and tricks

Summary

Highlights

• Learn the secret to mastering pelleting techniques in the lab like a pro 🔬✨
• Understand how different centrifuge rotors affect pellet positioning 🎯🧪
• Discover ways to safely separate supernatants from pellets without losing them ⚗️👍
• Find out how orienting your tubes can aid in pellet retrieval 🎯🔍
• Get step-by-step guidance on using decanting and aspiration methods effectively 🚰🔬

Key Takeaways

• Centrifuge techniques are crucial for separating components based on solubility and size 🤓🔬.
• Use fixed-angle and swinging-bucket centrifuges differently for best results 📍🔬.
• Retrieve pellets safely by knowing their expected position and using a consistent tube orientation 📐⚗️.
• Remove supernatants by decanting or aspirating without disturbing the pellet 💧🧪.
• Always keep both pellet and supernatant until you're sure of what you need 🧪🔍.

Overview

Pelleting tips and tricks Transcription

• 00:00 - 00:30 press the Appellate problems here's some tips for how you can become a superstar of the supernatant so a lot of times in the lab we like spin things down to pellet things out so we stick tubes filled with stuff into a centrifuge spin it really really fast and stuff that is insoluble and stuff that's large and like suspected and stuff um it's going to come out and into a pellet at the bottom whereas everything else is going to stay suspended in the liquid or dissolved in the liquid and we call that
• 00:30 - 01:00 liquid part the supernatant we can use this in order to separate things based on their like solubility and their mass and their size and things like that but once we separate them we need to like physically separate them like some separate that super name from the pellet and there are different ways to do this and sometimes you actually like lose your pellet or your supernated in the process and so it can be a pain so here are some tips for helping you do this successfully without like mixing
• 01:00 - 01:30 remixing them in the process or losing your palette when you're and not being able to find it in various things so for speaking of where to find it depending on what type of centrifuge rotor you're using your pellet will be in different places so if you are using um a fixed angle centrifuge where the tubes are held at a fixed angle what's going to happen is that the centrifugal force um where it's actually like pushing helping push the molecules it's going to make it so that those things
• 01:30 - 02:00 that you're pelleting out are going to end up like on to on the top Outer Edge so like at the bottom this like bottom corner type of thing um and so if it's in one of these tubes it's kind of going to be like a block here however you're using a swinging bucket centrifuge things that you the tubes during the Run are going to actually like be sideways um and so often we're using these when we're doing like um we have various centrifuges that we use with these swinging buckets and these are going to
• 02:00 - 02:30 go sideways what's going to happen now is that that force is going to push things to the very bottom when it's in a conical tube it's nice because you have everything kind of just like collecting this cone if you have a tube like this like when you're pelleting down cells trying to remove those cells from the media the food that they're growing in remove that media and keep the cells is often what we're doing and in this case your pellet is going to be on the bottom of the tube rather than like on the side of the tube sometimes it can actually be
• 02:30 - 03:00 kind of hard to find the pellet which is kind of seems that silly when you're talking about like pelleting down like cell culture um and you end up with these big gloopy glumps but you can totally see with your eye but other times when you're trying to peel it out really tiny stuff it can be hard we know where to expect it because of um if we know where to put which way we put the tubes in so it's helpful to always put your tooth in the same orientation so I typically put them so that the cap is when it's like I'm back for a centrifuge tube I put them all so
• 03:00 - 03:30 that the cap is like straight out um and I do this with all the tubes and now I know that I should expect the palette to be right here and what happens is when you take it out you can actually tell it's going to be a different size depending on how how much is there you can actually like outline your palette kind of like one of those like crime scene things like outline the palette so that you know exactly where it is um if you are dealing with a tube that um doesn't have like a line or thing you can just line it up based on the writing
• 03:30 - 04:00 on the tooth so that you know where to expect your palette um and then again for one of these it's not an issue though with the that's the one with the fixed angle with the swinging bucket it's always going to be on the very bottom and so that that's that um but you can still not need to mark it if it's a small pellet so then how you see the pellet and now your job is to get the Liquid off the pellet without actually disrupting um the pellet itself because well you just centrifuged it so that you can separate them and now if
• 04:00 - 04:30 you don't do anything they're going to mix back together potentially sometimes these pellets depending on what you're pelleting out they'll be really tight and firm and those are a lot easier to work with other times they're going to be all loosey-goosey and then it can be really hard to pour off this or take out the supernatant without actually disrupting the pellet um sometimes the pellet doesn't stick very well to the wall of the tube there are various things um and so you need to be careful so first you identify where the pillow is and now you need to get the Liquid away from it without
• 04:30 - 05:00 disrupting it so sometimes when you heat that liquid or that supernatant other times we're going to keep pellet either case sometimes we want both so you need to know what you're going after and make sure that you're not disposing of the supernatant if you need it or the Pele if you need it if you're not sure or if you you can keep both and it's always a good idea to keep both anyway until the end just to make sure that the thing that you that is the place where you thought it was so maybe it didn't
• 05:00 - 05:30 tell it out and so it's in the super neat still um and so if you keep that super neat and then you can test and see oh it is in the supernate so or maybe it you expected your protein to be soluble and still not liquid but what happened was it got insoluble and so it was with that holiday Gunk um and so you can test the pellet and see that it's in the pellet um so it's always a good idea to keep both um at least one practical and especially when you're doing an experiment type of experiment for the first time so how do you actually get that liquid off the SE way is like literally just
• 05:30 - 06:00 like pour it off um so this has worked really well if you have a really firm pellet and it's going to be fast so it's less the lesser of risk of the pellet and the super name like mixing that together just because they're like sitting there and things diffuse and move around um but you have the most you have a risk of actually like pouring out your pellet when you're doing it um and so you want to be really really careful when doing this um and that you're not actually going to lose the palette sometimes there's like a lid on like the
• 06:00 - 06:30 lip on here and if you do it carefully if there's if the pellet starts moving you can still like kind of capture it on the lip up here um and so this we call this decanting or just like pouring that liquid off um and so you can decant it off into a beaker or another tube or that sort of thing when you're doing this you're typically still going to need to follow it up with the next method aspiration um and sometimes you just start with the aspiration and aspiration sounds like this like scary word but really it's just like pipetting things off um so we can depending on how much
• 06:30 - 07:00 volume you're cutting off you might be with like a micro pack header it might be um with a pipette men with the serological pipette or it could be with an aspirating pipette which is basically like one of these except it's smaller typically like two mils or so um and it doesn't have a filter and since it doesn't have a filter what you can do is you can attach it to a vacuum line um and then actually suck it out um typically we're sucking it out into waist though so you wouldn't want to use this if you are trying to keep that supernatant these aspirating pipettes a lot like in
• 07:00 - 07:30 cell culture when we are removing media from like dishes of cells and things like that and then we can type out all that like Biohazard Waste into a waste container that gets bleached when you're using one of these aspirating pipettes with like the vacuum be really really careful that you're not actually touched you don't like actually accidentally like touch your palette with the vacuum because it's gonna suck it up hopefully it's gonna suck it up um so what can happen is sometimes like the tubing it's not like if you're at an
• 07:30 - 08:00 angle it's not actually going to suck it up well so you might have to like kind of suck it up and then move it suck it up and then like help it help help it out help that vacuum out depending on how good your vacuum is and things like that sometimes it can be easier to actually instead of using one of those long aspirating pipettes stick like a one mil tip um on the end of the vacuum line um and suck it up that way um but the basic idea is you just want to remove that liquid um and you want to remove it as quickly as possible so again you don't get that resuspension between the solid pellet and the liquid
• 08:00 - 08:30 supernatant often you're starting with one of these like bigger tools but it's not going to um do the full job if you use one of these tools that has a bigger that has a bigger tip so if like if you start with a one mil so that you can get up all the volume or if you start with one of these you can get up all the volume often there's going to be especially with when you're working with small volumes there's going to be a little bit of liquid left over so then you can take a smaller pipette which has a finer tip to get that last of the stuff this can be
• 08:30 - 09:00 really really important when you're doing things like those RNA and DNA extractions where things like isopropanol or ethanol might mess up a further step and so this is um it's important to get that elastic liquid and then also when you're doing the pouring off when you like pour it back now there's going to be a bunch of liquid probably or not a bunch but some liquid and then you can aspirate that liquid out so another thing to help with this prevent it recess spending by itself is when you're taking tubes out of the centrifuge leave them in the centrifuge like for one of those fixed angles leave
• 09:00 - 09:30 them at the angle that they're at um and as long as there's no one behind you like in line waiting behind you for the centrifuge just like taking it out one at a time after you take your pipette over if you're doing aspirating take it your pipette over to the centrifuge um and remove the liquid there like take them out one at a time remove the liquid carefully not disrupting the pellet and when you take your tubes out like hold them at the angle that they're at so the pellet it's not going to like move move because if you take your tube that's been like this and the pellet is like right here and now you take that tube like this and you
• 09:30 - 10:00 stick them in your tube rack well now what's going to happen is your that pillow is going to kind of migrate if it's not firmly stuck on there and it can start resuspending and so what you want to do is instead take the pellet take the tubes out like one at a time when you take it out hold it at the angle it was at remove that liquid um and dispose of that liquid and do this for each of your tubes individually um one at a time rather than just like sticking them all in your tube racking walking back to your bench unless people
• 10:00 - 10:30 are waiting um but hopefully there's no people waiting in line so once you aspirated the liquid now if you wanted that liquid if you wanted the super night and you can do whatever the heck you want with it um what if you wanted that palette um so there are different things that we do with this palette next but typically you're not just going to like leave the pellet in the bottle you're often going to like resuspend it so basically get it you took it out of one liquid and now you want to put it in another liquid so notice this is like a wash step so um or sometimes you do a wash without actually like Reese's bending the pellet
• 10:30 - 11:00 so you just like carefully like rinse it off without disrupting that pellet again um other times what you're doing is you're actually like resuspending the pellet in some other liquid um and then you either like spin it back down to remove that liquid and then you've resuspend it in something else that you wanted um because remember like that thing I was talking about where this can be stuff in the super name that you really want to get away from it and so the more of this time that you do this the more you're like diluting out that stuff that you're trying to get rid of because even though you've like headed out there's still a little bit of liquid always left over
• 11:00 - 11:30 um but so if you want the pal now you need to resuspend the pellet which is just like getting it into a suspension in another liquid um and the one that you like actually want so these is what what we typically do is we just like cut up and down um depending on the volume of your sample um yeah just like pipetting up and down um sometimes depending on how firm your stamp your pellet is and things like this it can be hard and it can need some help um so with like what we're doing like bacterial like pellets or whatever and
• 11:30 - 12:00 then we're trying to like wash our culture um often we do is like stick it on a Vortex there which like shakes it and helps it resuspend another thing if you have like a really small like really tight palette you can stick it in like a sonicating bath for a few seconds um and what we often do with uh ultracentric Beach tubes is if you have a pellet on here you can actually like scrape it against the um rack that's going to help break up the pellet into smaller pieces that then the water can do it or the liquid the liquid can like
• 12:00 - 12:30 do its thing and help it redissolve or resolubilize um or sorry like Reese suspense so we're talking about like things like when you're precipitating DNA or RNA during an extraction or purification where you take that DNA or you RNA and you add like isopropanol to get it to come out of solution so now it's undissolved but it's still like in suspension it's like floating around in your liquid um because these pieces are really really tiny um and they have like the buoyancy from the liquid and they have the friction and you're going to need to help gravity
• 12:30 - 13:00 out by applying the centrifugal force to push them out into your pellet but because this force is going to depend like the force that you need is going to depend on how big the thing is that you're trying to pellet out and this is why we can take advantage of different sizes and masses and molecules in order to like differentiate at least some different differential centrifuges them or whatever like separate them like the faster you spin the smaller the stuff that you can get out um to make it easier to actually get
• 13:00 - 13:30 these things out we often add like a co-precipitant um so with DNA and RNA we can add this thing called glycol blue which is glycogen that's attached to a Dye this is going to bind to that DNA and RNA um and it's going to help it get bigger so it can get pulled out more easily and because it's attached to a Dye it's going to look blue when you're actually going to be able to see it um so that can be really really helpful and even if it's not dyed it can still increase help it pellet and then increase the size of the pellet so you can actually see it
• 13:30 - 14:00 um in order to so those are the basic things um when working with pellets um so remember to find your pellet know where to find your pellets um so Orient your tubes in a way that you're going to know where the pellet should be look and see where the palette is to remove the super name to either by decanting um so pouring off by aspirating removing it with the pipette um and then do that with making sure that you're not actually disrupting the pellet and yet you do it quickly so that you're not actually allowing it to
• 14:00 - 14:30 resuspend um by itself