Spin column nucleic acid purification - how it works скачать в хорошем качестве

Spin column nucleic acid purification - how it works

If you’re looking for a spin class, have you tried out spin column DNA/RNA purification? I hope you don’t mind if my PCR products bind? Biochemists win when we take them for a spin! “Spin Columns” are a quick & easy way to purify pieces of DNA (or RNA) - just make sure you get the right kit! These columns are a form of “solid-phase extraction” where you bind the nucleic acid to a silica gel membrane, wash off the other stuff, then “un-bind” pure DNA or RNA. There are lots of different versions & kits & they work really similar so, although I’m going to be talking about PCR PURIFICATION (aka “clean-up”) the basic principles apply to other situations blog version (text old, video new): http://bit.ly/spincolumns ; YouTube: If you want to study DNA from cells, things are more complicated, because you have to break open (lyse) those cells, separate the DNA from all the other stuff (cell membrane, proteins, etc.) & make sure to get it really pure. More on how we use alkaline lysis (minipreps) to isolate whole plasmids here: http://bit.ly/minipreps For PCR PURIFICATION, you’re starting with a much purer sample, but you still have stuff you need to remove . What kind of stuff? PCR is a way to amplify (make lots of copies of) specific parts of DNA from larger parts of DNA. You specify the region you want copied using PRIMERS which are short fragments of DNA that bind to where you want the copier (DNA Polymerase) to start & stop.  It’s kinda like the original, bigger, DNA template is like a transcontinental railroad & you only want to copy the stretch from Utah to Colorado - the primers act as “train stations” that the DNA Pol “train” travels between, laying down “tracks” (DNA nucleotides) ahead of it as it goes based on the sequence of the other strand. Lots more about it here: http://bit.ly/pcrtrain For now, let’s just think about what that train needs – it needs the primers (stations), nucleotides (train tracks), & it also needs salts to keep it happy, & it especially needs magnesium (Mg²⁺), which helps it hold & coordinate the addition of more nucleotides So you do the reaction & it makes lots & lots of copies, but now you need to separate those copies from that other stuff. We commonly do this using spin columns which have a silica-based membrane. The premise behind these is: mix reaction mix with stuff that will denature (unfold) the DNA Pol & make big pieces of DNA bind the column, but allow everything else to flow through. Wash it to make sure you really do get everything else to flow through. Then change DNA’s environment so that it unbinds & goes through The key is to find conditions in which the DNA would rather bind the solid membrane than the liquid & conditions in which the situation’s flipped – the DNA would rather bind the liquid than the solid.  Let’s look at the players. The membrane in silica-based. silica (amorphous silicon dioxide, SiO₂) can have a lot of modifications & the composition of these membranes are proprietary, so I don’t know exactly what their surfaces actually look like at the molecular level, but silica has hydroxyl (-OH) groups that at low pH (acidic, where there are lots of free protons (H⁺) floating around) will be protonated (-OH) but at higher pHs (where there are less free protons) will be deprotonated & thus negative (-O⁻). In addition to this group, silica also offers up some additional opportunities for hydrogen bonding & even hydrophobic interactions.  & the DNA? DNA’s a polymer (chain of similar repeating units) of nucleotides (DNA letters). These have a generic sugar-phosphate backbone they link through (same-strand bonds) & unique nitrogenous bases (A, T, G, or C) that stick out & form the basis of between-strand base pairing.  The phosphate in that sugar phosphate backbone is negatively charged, & it’s really happy being negatively charged, so even at pretty low pH it’s gonna stay that way. “Normally” the nitrogenous bases are neutral. BUT, as the name suggests, they are (weak) bases, meaning that if the pH is low enough (meaning there are lots of H⁺ around) they will pick one up & this makes them + charged. So, at lower pH, the overall charge of DNA decreases, so it becomes less water-soluble.  This helps us separate DNA from RNA in liquid-phase RNA extraction. At a low pH, DNA prefers the organic phenol-chloroform phase, whereas RNA’s extra -OH keeps it in the aqueous phase. http://bit.ly/rnaextraction Here, at a low pH, DNA doesn’t have an “organic phase” – instead, it binds (adsorbs to) the silica membrane. Note: I’m not sure if the pH is low enough here to change those bases, but there’s another important function of the pH. At a low pH, the sometimes OH, sometimes O groups of the silica are more likely to be in the -OH form, so there’s less negative charge that could repel the negatively charged DNA backbone. finished in comments