PCR purification basics? - (Aug/01/2005 )
Hi, I'm new, and I posted this question up in the "pinned" topics, but maybe I should be starting a new topic for this? I don't know. Here's my question:
"This is a silly question, but I was just thinking today about how I use the Qiagen PCR purification kit all the time, but have no idea how it works! What do the buffers PB and PE do? I guess PB helps DNA bind to the spin column, but how? And why do you need to add ethanol to buffer PE? What does it do? Thanks so much."
may you give the composition of these buffers? And the step they're used...
I think the exact composition of their buffer is proprietary, I fear. They don't want you to know how it works, because you'd be able to 'build' the same kit for a tenth of the price...
You'd still need the columns, though. A lot of Qiagen's buffer compositions are available if you call them up and ask.
I do know that PB contains chaotropic salts, which alter the structure of water. These salts are capable of disrupting protein structure by destabilizing hydrophobic interactions, but have little effect on DNA and other polymers. So PB, I think, is supposed to get rid of DNA-binding proteins and other contaminants (in this case, polymerase).
As far as PE goes, I think it's supplied as concentrated buffer + salt, to which you have to add ethanol. DNA precipitates out of 70%EtOH, so it serves as a useful wash to remove other components. It basically just prevents DNA from dissociating from the column while you wash away other contaminants (salts and such).
In this case, though, I don't think it's quite as important. Quiagen claims that DNA binds their resin under high-salt conditions regardless of ethanol content. Maybe it's just to make the solution more volatile and easy to remove? Could also be a throwback to the "old" way to purify DNA via alcohol precipitation - it worked in the first trials and they never saw the need to exclude it. Might also serve to remove primers (don't know how short stretches of RNA behave in those conditions).
Thank you for your replies.
All I know is that buffer PB contains high salt, which somehow helps DNA bind to the column.
Buffer PE contains ethanol, and is supposed to wash away dNTPs and primers and other things that aren't your PCR product. In this case, I don't know why the PCR product, dNTPs and primers don't all precipitate out of the ethanol solution...???
The elution buffer contains low salt (apparently you can also use water), which somehow elutes the DNA from the column.
I still don't really understand the "why" behind all of this, especially the ethanol part. Thanks for any help!
I can help out a bit.
The first buffer, the high salt one, is the one containing the Chaotropic salts (e.g. NaClO. and others like it.) So they're big and toxic. the chaotropic salts are used to alter the characteristics of the water molecules which surround the DNA molecule. In this way the positive H cloud is weakened so that the DNA molecule can more easily bind the the Si-based matrix.
The washing buffer contains non chaotropic salts and the alcohols. The alcohols are use to "precipitate" the DNA more firmly to the matrix. Since in general your DNA molecules of interest are much larger than your primer dimers they have more binding positions om the matrix. The more important reason for incorporating the washing step is to get rid of the chaotropic salts cause these things wil inhibit just about anything PCR,Qpcr and even the new generation of ABI sequencers can't always handle these kits.
This principle has been patentend bij Boom and you should be able to find a detailed descrpition of the procedure with a internet search. As far as i know all commercial companies with column based kits use this technology (Qiagen, Machery-Nagel,Sigma, Promega, Clonetech,Takara, Gentra,...) So far the ony company with columnbased kits who have been able to develop their own technology is Invitek. They use 100% chaotropic free kits.
hopes this helps you out.
You will need to know the principles behind this kit for many protocols. The high salt decreases the negative charge on the DNA allowing stronger interactions with the column membrane. The low salt increases the repulsion between the two, allowing elution. Look at an RNa extraction kit to get a better understanding of the principle (I hope I have got it the right way around) The manual usually tells you roughly how it works though. Quiagen manual does.