2
$\begingroup$

In the Materials and Methods section of this paper on DNA extraction and analysis from hair I do not understand some parts of their protocol methods. In particular:

"The digested hair solution was then extracted twice with two volumes of phenol saturated by Tris-EDTA (TE) buffer (Sigma), and once with two volumes of chloroform. "

How do the volumes of phenol and chloroform come into play? I know they are used for an organic-solvent extraction, but what was the actual procedure used, i.e., the steps for adding each chemical?

Another question I had was on the buffer they used:

"0.01 M Tris-HCL(ph8), 0.005 M EDTA, 0.1 M NaC1, 0.039 M DTT, 2% SDS"

That whole combination would be the "Tris buffer" then yes? And the proteinase K would then be added to some of this buffer to digest the hair?

| improve this question | |
$\endgroup$
  • $\begingroup$ http://www.bionut.ki.se/groups/mer/doku.php?id=mouse_tail_dna_extraction You might want to check this protocol for phenol/chloroform extraction. It is tricky to say which is Tris buffer, but to digest hair, proteinase K would be added into buffer containing Tris, EDTA, NaCl, SDS and DTT. However, I feel 2% SDS is a bit too high. Check the protocol again. $\endgroup$ – 243 Jun 9 '15 at 1:31
5
$\begingroup$

The DNA solution has a fixed volume (e.g., 0.1 mL). To a suitable test tube is added 0.2 mL of buffer-saturated phenol. After closing the test tube the mixture is mixed well, typically using a vortex mixer, but possibly by careful, repeated inversion. The aqueous phase and the organic phase are barely miscible. The tube is centrifuged to speed up the equilibration, and concentrate the denatured protein at the interface. After centrifugation the aqueous layer is carefully removed to a fresh test tube.

That is one organic extraction. This is repeated with a fresh aliquot of buffer-saturated phenol (another 0.2 mL). Then the aqueous phase is decanted again and extracted with 0.2 mL of CHCl3.

According to the recipe you provided, the buffer-saturated phenol was purchased from a vendor (Sigma-Aldrich Chemicals, of St. Louis, Missouri, USA), so you can look up the ingredients and composition on their online catalogue (no doubt).

The buffer recipe you provided is the proteinase K digestion buffer. It is typically performed at an elevated temperature.

This protocol is designed to isolate DNA from the nuclei of hair cells. Fully half of the contents of a eukaryotic nucleus are chromosomal proteins, by mass, so the proteinase K digestion step not only breaks open the hair cells, but also helps to remove all of the unwanted, contaminating cellular and nuclear proteins.

| improve this answer | |
$\endgroup$
  • $\begingroup$ How does the cholorform come into play though? $\endgroup$ – Ro Siv Jun 9 '15 at 1:45
  • $\begingroup$ Chloroform is also an organic reagent. It forms a dense immiscible layer and helps to remove any residual phenol from the aqueous phase, and also should clean up the last traces of denatured protein. You should consider obtaining a biochemistry laboratory manual that explains these basic techniques. $\endgroup$ – mdperry Jun 9 '15 at 1:53
  • $\begingroup$ Would you have any suggestions for a biochem lab manual? $\endgroup$ – Ro Siv Jun 9 '15 at 2:22
  • $\begingroup$ @RoSiv Sambrook & Maniatis Molecular Cloning is a good start. $\endgroup$ – March Ho Jun 9 '15 at 3:49

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.