FloodLAMP Test Training Video Course
Video 05 - Reaction Mix Prep

Preparing a reaction mix for 48 reactions, including handling master mix and PGS, and logging procedures as per SOPs. Provides detailed instructions on preparing strip tubes, avoiding contamination, mixing and measuring components correctly, and storing the prepared mix in the freezer for future use.


Test Trainer  [0:00](https://vimeo.com/668261400/fa2b78d5e7?ts=0)
Okay, so this video is going to be to show making the reaction mix and we're going to make enough for 48 reactions. That's how the PGS comes packaged for 48. So you're going to use this whole tube And then this is a new master mix, so we're going to end up using half of this guy. There we go. So these have been thawing about 10 minutes. So if you plan ahead, then you have to hold them less to get them to finish thawing and you sort of want to see. This guy's not quite thawed. That guy not either, but that's okay because I need a few minutes to just get sorted on my strips. So I'm gonna put these into strip tubes. You could also do a plate. I'll show a little bit about that at the end. So I'm going to get out my way, I like to keep the strip tubes in this kind of container. It's just easier to get out. And I'm not going to be marking them. I mark them when we get them out and use them. It just saves time and sometimes you cut them in half. Okay. Trying to Make sure you don't touch the inside as you do this. I don't touch the ends that are sticking out. As soon as you get them done, close that. So, here's our sign for making the reaction mix. It has the set up for eight and for 48. If you end up wanting to do a whole plate 96, you just do 2 48s and add them together. If we look over here at the long run form, and this says mini system but it would be the same, it says to start by writing down the entry on the log sheet prior to starting the prep. So we'll go ahead and do that and show you what that looks like. So here's our reaction mix SOP. I should say, as you can see, we have another entry already. Now this corresponds to this lot number for the master mix, and it's the same lot number. So we don't have to do anything new here. If you end up using all of one line, you switch to another one, then you can retire this form and get a new one. So we're going to just fill this out. 11722 3pm. It's going to be RM_0117-1. And then this is going to be a new master mix ID. So we'll call this one underscore 0117. We do like to identify these guys uniquely. So you'll want a little towel to wipe these guys off so they're not so wet. And to this one, we're going to use up. This one we can write 01 17 there we go So now we know which one we're talking about. And it helps, too, to see that on the cap. It shows that we have used it. So the PGS here, yours will probably have a lot and an expiration. This one's a little bit older, so it doesn't have that. But it says PGS underscore 210604. So same thing, and it has that same expression. And then this is the key part. We're just going to write down what we're doing here. If I'm doing the whole thing. So we have two entries here, kind of just spelled out for you, either 8 reactions or 48. If you're doing, say, three strips instead of just one strip of eight, then you just multiply these numbers by three. If you're doing a whole plate, you just do two PGS tubes. *And that's because this is set up so that the amount is is correct for adding 655 straight to this tube you don't need another intermediate tube.* If you were doing a strip of eight or some intermediate amount you would want to get one of your 1.5 mil tubes and use that and go from both of these into this tube as your as your reaction mix too. Okay so the key here is 655 that's the number in the set. It's going to be the big pipette, P1000. Go down to 655, get ready. 655, there we go. Okay, we've got to get one more thing to be ready. Trough. Actually, we've got a few more things to get ready, but this is one important one. Trough out. Always make sure to have your foil handy. Piece of foil. Pull this guy out. Now the thing that we need is our multi-channel pipette over here. So we're going to have this guy ready for when we're ready to go. Okay, so let's check our reaction mixes. Oh, yep, See it's liquid now. And you want to look at this one to see if there's any ice. Doesn't look like there's any more ice. So you vortex you guys together for about 10 seconds. Okay. Then you pull out the ballast tube here. Put these in opposite. Give this a quick spin. That's all you need. You're just trying to get it from the top. There we go. Okay. And you do want to move reasonably quickly through this so that these guys aren't sitting for a long time. And in fact, you know, I would recommend, especially the first time you do this, to go ahead and get a cold block out. In fact, you know what, I'm gonna just go ahead and do this because I'm gonna freeze these guys afterwards. I'm not gonna use any of them. So normally you want to get your cold block out about 10 minutes before so that it's not freezing. Actually my freezer is set a little bit warmer than standard, so it may not freeze anyway, but I'm going to go ahead and put these in. I'm going to keep this rack handy because this is what I'm going to put them in afterwards. Okay, this guy's here. Keep that here. And then you do want your lid handy too. This lid's a little bit bigger, so it fits nicely. Okay, I think we are ready to go now. So we need to add the 655 to this guy. Open this up. So this is the master mix here. This is new, it's tight. It's the same thing with all these screw cap lids. You want to do that little bend so that they don't flick. You want to be careful here not to stick the pipette or too far down in there when you start because otherwise you'll overflow the tube. Make sure you get the nice full tip. Beautiful color. There you go. Tip touch on the side. There we go. I'm just going to go ahead and get this guy back in the freezer so I don't have to worry about it degrading. Okay give this guy a little pulse and then we're gonna stick him in here so we get all liquid down. You don't want to run short. Okay. There we go. Now we're just going to use this same setting to get it into the reservoir. Oh, don't need that open yet. Okay, So here you want to try to be careful to not create too many bubbles. So I'm just going to go pipette in like that. Oh, so you got a bubble there. Don't worry too much if you do. Okay, there you go. Not too bad with the bubbles. This guy just goes in the trash. Now your multi-channel should just live at 23. That's how much you do for each one. So, there you go. Okay, see, we popped those bubbles. I like to balance this one. I go all the way to the bottom and no blowout. Just be slow and steady. Just be slow and steady. Number three. Number four. Number five. Last one. The last one can be the trickiest because you want to make sure all your tips are full. So if your table's tilted, you can have a problem. If you've created too many bubbles, you can have a problem. And if you do have that problem, What I would recommend you do is go ahead and just get two tips or four tips or whatever you need in order to get you know You need to get some more you can get some more like that See you get a few more reactions if we needed. There's a little bit of overage here. So this is a nice little trick to know. I'll just put this back in here. Okay, so now all we gotta do is cap our tubes and get them in the freezer. 48 reactions worth. So in terms of capping these guys, again you want to make sure to not touch the inside of the caps. So see I'm only grabbing it by the edge there. What I like to do is just kind of press it on like that. I'm gonna come back and press them all down. I think sometimes I move them before I put the caps on because you press hard into the... See how they do on this rack. They can get stuck on these PCR racks. If they do, you just push from below. That's how you kind of get them unstuck. OK. Just avoid touching the other strips. OK. So Now I'm going to come back through here. All nice and clicked flat. There we go. If you don't get this part right, they will turn slightly orange and then you can't use those. Whichever tubes turn orange you can't use. So I'm going to come back a second pass to make sure they're nice and flush. Okay. There we go. Get this guy out. Yeah, they don't get stuck on this. You want to inspect each one as you pull it out. Looking really good. I'm actually going to put them over here. These are all the same, so there's no particular order to them. There we go. Nice looking strip tubes. They're all ready to go. You can do 48, you can do 96, you can do 192, as many as you want. So we need to do a few cleanup things here. We are going to make sure that we label this with the ID here 0117-1. There we go. So I like to just put this, recommend just putting this on the top. You want labels that stay on in the freezer. So if you don't have any, you can recommend some. Okay, there we go. Then finally, put this guy in a Ziploc bag. You don't want to jostle it too much because you want it to freeze with the reaction mix all just down at the bottom. So there you go. Into the freezer. Thanks for watching!