Lab 3: PCR Purification transcript

hello everyone welcome back for this week's experiment we will be doing a pcr product purification and restriction enzyme digest you can follow along with me in your lab manuals on page 18. so remember last week we ran a pcr and we obtained our pcr product for the beta gal gene so now we need to purify all of that all of our beta gal gene away from all those free nucleotides and salts and other cofactors that were in that mixture so that is why we're going to run this pcr purification uh the kits that we'll be using to purify our dna is this nucleus spin gel and pcr cleanup so to begin i already have in this tube 80 microliters of binding buffer this buffer will help our pcr product to bind to the silica gel in our spin column and i'm just going to mix in 40 microliters of our pcr product so this should give us a total volume of 120 microliters i'm going to make sure to mix thoroughly all right so once we have our pcr products thoroughly mixed i'm going to transfer the entire volume into a silica spin column so right here you can see is our silica spin column it's detaches into like a waste tube and this is our actual column so if you look closely you can see that the white underneath that yellow ring is the actual silica so that is where our dna will bind to so i'm just going to draw the entire volume inside my tube and just transfer it into my silica column all right so the next thing i'm going to do is place it into the centrifuge and make sure to counterbalance it with about with an appropriate volume so remember it was about 120 microliters so this is just a counter balance sorry about that forgot that i needed a top so i'm just going to place this top down close the centrifuge and we're going to spin it so we're going to spin it at max speed so 13.4 rpm for one minute so as the centrifuge spins it'll draw all the liquid out of the silica column and as it pulls the liquid down the protein will get stuck inside of the column all right so now that our column was able to spin for that one minute i am going to remove our column and you can see down here that all the liquid has transferred to the bottom of the collection tube so the next thing i'm going to do is add in 200 microliters of wash buffer to the column and we're going to make sure that we do this wash twice so here's our wash buffer remember this wash buffer contains ethanol to remove all the residual salts that might be stuck to the column all right let me make sure the counterbalance is correct okay and we'll do it at for the same amount of time at the same speed so i'll just do this for another run and i'll get back to you guys after we are done with the wash step all right so now i have completed washing our column so you can see that we've washed it about two times this time a ton of liquid collected at the bottom of the collection tube so the last thing we need to do is just remove our column from the collection tube so our dna should still be in this column and we're actually going to put it into an empty eppendorf like so so now what we're going to add is 35 microliters of elusion buffer this elusion buffer will allow for our dna to solubilize and exit the silica column so i want to make sure that when i add the illusion buffer i add it right at the center of the silica matrix so i want to let this sit for about a minute this way i can allow for the buffer to kind of help solubilize as much dna as possible so we can just collect all of it it'd be a shame if we just let it we just put it in and immediately spin it down and we only collect about a third of the dna we were supposed to so just let that sit for a little bit all right so a minute has now passed and i'm going to add it into our centrifuge make sure that our counterbalance or counterweight is the appropriate weight all right make sure that they're as far apart from each other as possible and just like all the other steps i'm going to spin it at 60 seconds at 13.4 rpm all right so we've finished up here purifying our beta gal dna so you can see in this tube is our 35 microliters so now what we need to do is start our digestions so we're going to be digesting both our pure beta gal gene as well as this pet 30a vector which will be the plasmid that we attach our beta gal gene into so i'm going to be using the same 35 micro leads i guess the entire 35 microliters of our beta gal dna and into that i'm going to be adding 8 microliters of water all right then five microliters of 10x buffer that will help the digestion occur a little more properly so here is our 10x buffer and the last things i'm going to add our are our restriction enzymes so remember that these are enzymes so they are proteins we have to keep them on ice or else the restriction enzymes might lose their function so this will be one microliter of our first restriction enzyme hind3 as well as another one microliter of our other restriction enzyme bam h1 so with that all together in that tube or in our digestion should be about 50 microliters i'm just going to leave it in the ice and the next thing i'm going to do is start setting up the digestion of our plasmid or the pet 30 vector so for this one i'm going to start by adding 35 microliters of water or 33 microliters of water sorry 33. into this empty tube and next i'm going to add 10 microliters of our actual vector so the pet 30 vector so remember because each microliter has about 1 microgram that means i'm going to be using about 10 micrograms of pet 30a mix that in next i'm going to add that 5 microliters of the 10x buffer and then again the last two components will be our restriction enzymes so starting with i guess bam h1 and then hind3 so with that we now have both our digestions ready to go we just need to make sure that we incubate them at 37 degrees for about 20 minutes this will allow for the reaction to occur and so once that is done i will start setting up the gel for the gel extraction all right so it's been about 30 minutes since the incubation both our beta gal as well as our pet 30 digestion are now ready to go into this gel so you can kind of see that there's already things inside the gel because i already added everything in but something happened with the recording so i'm going to kind of explain the process once more so in the very first well we have just like last week a molecular ladder so we can compare our pcr products as well as our plasmid digestion to this ladder to determine if it's the right size in the very first well i have about five microliters of our pure beta gal gene that we digested mixed in with five microliters of an orange an orange diet so that you can see it inside of the gel and in the second lane or i guess in the third lane i have the digestion for our pet 30 so 5 microliters of this with another 5 microliters of our actual of the orange diet so i added both of these into these two wells and i'm just going to let them run for about 30 minutes at 120 volts so very similar to last week so from this gel what we're mostly trying to see is that if our stuff was actually digested and this will be useful for us in the future so that when we know that it's adjusted we can actually combine these two and ligate them so that we can actually have a plasmid with our gene in the middle so i'm just going to run this gel and that'll be pretty much the end for this week's experiment