Hi Akila,

There are a lot of literature available that will give you more details so I will just be very brief here. Guanidine HCl is a strong protein denaturing agent that will denature the protein initially to release the nuclei acid and NaAc serves to maintain the pH at this point.

I don't think you are isolating the RNA at this stage, you are just trying to retrieve the whole nuclei acid. RNA is usually isolate from the whole nuclei acid later, using LiCl precipitation for example. If you are using a kit, the column might be treated to specifically trap RNA. Or if you are using PCI method, using phenol at a pH of 4.5-5 will allow for isolation of RNA. Whichever method you use, you will probably have to DNase treat the extracted RNA in any case, to eliminate DNA contamination.

Hope this helps : )

Thank you very much Su San, yeh I've used silica milk and wash buffers further to isolate RNA after this Extraction procedure.

I've got another clues about NaAc;

+ NaAc has nothing to do with stabilizing duplexes but provides moderate concentrations of monovalent cations which is required for DNA to precipitate.

+Cations can neutralize the negative charges carried by phosphate groups, which are important for solubility.

+Further more it increases the competition between salt and DNA for hydrate water.

+The purpose of adding salts is to neutralize the charge on the sugar-phosphate backbone of the DNA. For a precipitation, you need to form ion pairs between the polyanion (DNA) and the cation (usually sodium or ammonium). The DNA and counterions are more or less in the free ion form rather than the ion pair form in dilute aqueous solution, and are surrounded by one or more layers of water molecules. Adding ethanol decreases the dielectric constant of the solution. The anions and cations form ion pairs and this results in precipitation of DNA.

~Also thanks should goes to memari & bob1 of bioforum too.

The term "chaotropic" means chaos-forming, a term which in biochemistry usually refers to a compound's ability to disrupt the regular hydrogen bond structures in water. Hydrogen bonding profoundly affects the secondary structure of polymers such as DNA, RNA, and proteins, as well as how water-soluble a molecule is. Under native conditions, nucleic acids are covered by a hydrate shell consisting of water molecules that maintain the solubility of DNA in aqueous solutions. With the addition of chaotropic ions to the nucleic acid, this relatively ordered structure of water molecules of the hydrate shell is destroyed.

The chaotropic salts create a hydrophobic environment. Under these hydrophobic conditions, the silica membrane of the NucleoSpin columns is the most suitable binding partner for the nucleic acids. Proteins, metabolites, and other contaminants do not bind to the membrane and therefore are washed away during the subsequent washing steps.

As a further feature of the chaotropic salts, the respective cations saturate the silica membrane with positive charges, which still improves the binding of nucleic acids under hydrophobic conditions. Chaotropic salts increase the solubility of nonpolar substances in water.

They denature proteins because they have the ability to disrupt hydrophobic interactions. They do not denature DNA or RNA. Their function in the NucleoSpin Extraction Kit is to denature cellular proteins (such as DNase and RNase). The high concentration of salt also facilitates binding of the nucleic acids DNA and RNA to the silica membrane in the column.

~ From: http://wiki.answers.com/Q/What_happens_to_DNA_in_the_presence_of_a_chaotropic_salt

Dear Akila, 

Su answer is excellent one and what you wrote above is also true  but maybe you will find also my paper interesting;

https://www.researchgate.net/publication/261798279_The_Adaptation_of_Silica_Capture_RT-PCR_for_the_Detection_of_Potato_Virus_Y

You may find there references to the action of chaotropic salt on nucleic acids.

Article The Adaptation of Silica Capture RT-PCR for the Detection of...

this link is useful

http://qa.answers.com/Q/What_happens_to_DNA_in_the_presence_of_a_chaotropic_salt

regards

Check this link

https://www.quora.com/How-is-DNA-affected-by-the-presence-of-a-chaotropic-salt

regards

I have a question.. Why is guanidine thiocyanate used for agarose gel solubilization (during the DNA gel extraction purification procedure) as opposed to guanidine hydrochloride?

after solubilization of the agarose gel, couldn't ethanol be used to precipitate the DNA? If my DNA sample is already very pure, I don't see what advantage there is to capturing the sample with silica gel resins?

Isaac

Hi Isaac. Ethanol would precipitate not only DNA but also agarose. It is why you need to separate these two poly-sugars by another approach.

Concerning your first question, maybe the answer is that the thiocyanate is more powerful chaotropic compound? Definitely, it is beneficial to promote DNA binding to silica. In the past, I have been using for the same goal another chaotropic salt - potassium iodide.

Hi

Usually Buffer PB contains a chaotropic salt which promotes DNA absorption to silica. Hope it help!

Would it be necessary to use chaotropic salt in the homogenization buffer for DNA/RNA extraction in the presence of Prot K? what is the added value of chaotropic salt?

Dear Chopie. Not. Proteinase K, as any other protein, will indefinitely denature and won't digest anything in the presence of chaotropic salts. That is why these salts are so good at inhibiting nucleases, including ribonucleases. Therefore, these salts are beneficial for the purification of intact RNA. Another advantage is that in the presence of these salts, nucleic acids adsorb to polyols on the surface of materials like cellulose, silica (including glass beads), or some magnetic particles. This phenomenon is responsible for the magic lying behind the homemade and commercial kits for DNA/RNA purification in the column format (in case of silica) or using magnets.

If you use protocol involving Prot K and want to modify it by including chaotropic slats, you should add them after Prot K will do its job.