Centrifugation

[Melstmu2010]

Hello!

I have a question regarding Polymerase Chain Reaction and centrifugation. The protocol I am using requires centrifugation during the isolation procedure. Specifically, I centrifuge for 10 minutes at 14,000 rpm after adding phenol: chloroform: isoamyl to sample DNA. My question is, why is a speed of 14,000 rpm necessary? I've been told that it is not necessary in all protocols, but i'm asking why it would be necessary for this protocol.

Thank you!
Melanie

[JackBean]

because that shortens required time and improves separation

[Melstmu2010]

How does it improve separation?

I'm presenting the entire process of PCR to my boss and no answer seems good enough for him (based on people that have presented in the past). He definitely wants a description as detailed as possible, but i'm not sure what he's looking for exactly.

[canalon]

Okay,

the DNA isolation and the PCR process are actually completely separate. You can purify Nucleic acids for plenty of other things.

The point of centrifugation of the mix during the phenol chloroform step of the NA purification is to separate the 2 phases of the solution (aqueous containing the NA, and anhydrous containing most of the phenol and some of the proteins). The faster you go, the easier and faster the separation will happen. But 3000rpm for a few hours might work as well. The point in this step is to get rid of the denatured protein (at the interface) and the phenol which will mix better with the chloroform than with water, and which could be a big problem if it stayed mixed your NA for whatever you would want to with them later on (like PCR).

[Melstmu2010]

Thank you!