Spin column nucleic acid purification

Spin column nucleic acid purification - how it works

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@thebumblingbiochemist Жыл бұрын

It’s not just the low pH that’s important. You also need salts. CHAOTROPIC SALTS like guanidinium hydrochloride bring “chaos” to water -> they disrupt water’s bonding networks, “loosening up” the water coat surrounding the DNA so the DNA can seek out & bind to the silica instead. When it does so, you get an entropic benefit because it frees up water molecules that were stuck so they can move around more, thus increasing entropy (disorder)more here: bit.ly/ionicstrengthsalting There are different ways it can bind, & the actual situations probably a combination of lots of them. The exposed bases could form direct H-bonds or hydrophobic interactions with the silica; Cations from the salts could form “bridges” between the backbone & the silica; etc. The more interactions, the stronger the binding. The smaller DNA pieces (primers) don’t offer enough binding opportunities to stick well, so they flow right on through with the other stuff So we start by mixing our (completed) PCR reaction with a solution containing guanidinium hydrochloride (the chaotropic salt to loosen the water shells) &, to help get the DNA to precipitate (come out of solution), ISOPROPANOL (aka isopropyl alcohol, aka propan-2-ol, etc.). This lowers the dielectric constant -> reduces electrostatic shielding so that the salts, DNA, & silica can find each other. & it “dilutes” the water, so there’s less water available to bind the DNA, so the DNA “dehydrates” & really sticks on tight to that silica so it doesn’t come loose when you do the washes. If you’re using a QIAquick PCR purification kit, this is buffer PB. (This isn’t a paid ad for Qiagen or anything, it’s just what our (and many) labs use). The kit also includes a pH indicator you can (& should) put in! (unless you’re gonna be using the DNA for a super-sensitive microarray) - you want to make sure that the pH is low enough (below 7.5). If the solution is orangey-purpley, it’s too high & the DNA won’t bind well. Don’t worry, just add a little acid (sodium acetate, pH 5 does the trick) -> should turn yellow Now you need to do the actual binding part. The silica gel membrane is held in a microspin column you can transfer between tubes to collect the flow-through & you pipet your sample into the “cuppy” part above the membrane. Then you centrifuge it (spin it really fast) to pull the liquid through. (alternatively, if you have a lot of samples to do at once, it can be quicker to use a vacuum manifold which sucks it through (though you’ll still have to switch to spin for the elution because it sucks it through into the waste…)). The bigger pieces of DNA (>100bp) will (hopefully) bind the membrane, but all the other stuff (primers, denatured DNA Pol, salts, etc.) will flow on through & you can toss it (the liquid, not the column!) Now comes the wash. Now that you’ve gotten DNA stuck on there tight, you want to wash off any lingering extra salts. You do this using a buffer containing ethanol (buffer PE in Qiagen’s kit). just add the liquid the same way you added your sample, spin it through & toss it out. DNA’s not soluble in ethanol, but salts are, so the extra salts are removed. It’s really important that you remove that guanidinium chloride because, while you wanted to denature the DNA Pol, you don’t want it to stick around & interfere with any reactions you do with the DNA later on. After the wash & toss, spin it again to give any residual ethanol another chance to get pulled through. It’s really important to remove the ethanol or the DNA will have problems dissolving &/or when you go to load samples into an agarose gel (more here:bit.ly/2Z6vmeR) they’ll float up out of the well cuz ethanol’s less dense than the buffer. Now that you’ve gotten the DNA clean, you need to get it to unstick from the silica. To do this, you need to reverse the conditions that got it to stick. We get it to stick with high salt, low pH -> we get it to unstick with low salt, high pH. The Qiagen kit comes with an elution buffer (buffer EB - which is 10 mM Tris·Cl, pH 8.5) (Tris is a buffering agent) you can use or you can use plain old (but really pure, nuclease-free) water. If you use water, give it a minute to fully dissolve the DNA before you spin it through. more on storage buffers: bit.ly/oligo_working ; KZbin: kzbin.info/www/bejne/bIfFdWWPZ6-Ji8U Speaking of which -> once you redissolve it, you spin it through into a NEW TUBE - this one you want to keep! Then, take your spun-through, pure DNA over to the spectrometer like a NanoDrop & check out its absorbance spectrum (it should have a 260/280 ratio of ~1.8). More on that here: bit.ly/dnauvbeer more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com

@benjamindiazzamora5048 Жыл бұрын

Hello, my name is Benjamin, I am a geneticist and biotechnologist. I love your videos, you inspire me to continue researching. Many blessings!

@thebumblingbiochemist Жыл бұрын

thanks so much! best of luck with your work

@doviende Жыл бұрын

thanks! as someone new to genetics, i really appreciate the detail you've put into this. a lot of intro resources just say sort of "here are some steps", but i really like that you've included a bunch of the background reasoning.

@thebumblingbiochemist Жыл бұрын

Thank you! That's exactly what motivates me! (well at least a main part) I'mm really glad you found it helpful and wish you the best of luck in your studies/research/whatever you're doing!

@thmhlckdio.01 5 күн бұрын

Thanks to your video, it's a good source for me to do my presentation!!!

@thebumblingbiochemist 5 күн бұрын

Glad it was helpful! Good luck!

@sekayitigere 9 ай бұрын

Thank you for this. This is really the best explanation I’ve had on this 🙌🏾🙌🏾🙌🏾

@thebumblingbiochemist 9 ай бұрын

Thanks! So glad it was helpful!

@teguhsantoso5248 Жыл бұрын

Thank you. Its complete my understading of purification DNA methode.

@thebumblingbiochemist Жыл бұрын

Glad it was helpful!

@basilbaby1577 8 ай бұрын

Could you explain the purpose of using preheated water (70°C) for nucleic acid extraction ?

@thebumblingbiochemist 8 ай бұрын

I assume that's just to aid the dissolving

@mn-mw2pc 10 ай бұрын

What the purpose of adding proteinase enzyme together with lysis buffer in DNA extraction? Thanks.

@thebumblingbiochemist 10 ай бұрын

Helps break down the proteins so they don't co-purify with the DNA and/or cause the DNA to get precipitated out with the protein

@vibriofischeri4507 11 ай бұрын

So do you use guanidinium thiocyanate or guanidinum chloride? I didn`t get it because on picture it`s guanidinium thiocyanate, but later you talk about guanidinum chloride. And thank you for a great video! You explain biochemistry really cool!

@thebumblingbiochemist 11 ай бұрын

Different protocols use different ones, sorry! The guanidinium is the key part, but the counterion can vary

@vibriofischeri4507 11 ай бұрын

@@thebumblingbiochemist Thank you for such a rapid answer! And thank you so much for the great video. It's really the most precise explanation of the principle of DNA purification I've found on the whole internet!

@thebumblingbiochemist 11 ай бұрын

thank you!