genomic dna isolated from blood is brown color?! - (Nov/22/2009 )

Hi, i isolated mouse genomic dna from blood using traditional methods as follow: Lyse cells, add proteinase k, phenol:chloroform:isoamyl extraction, chloroform isoamyl extraction, ethanol precipitation.. The end product of genomic dna i have is brown color. I believe its the pigment from the RBC but does anyone know how to remove it?

I tried doing another phenol:chloroform:isoamyl but its still dark colored. Please help!

krystle on Nov 22 2009, 08:09 PM said:

Hi,

I usually do dna purification from blood using Qiagen kit. I have the same problem when the blood is very old and the purification and washing don't work fine. So what you can try is to wash again with ethanol.

I don't know for what you need the DNA, but for some procedure this is not a problem ( I use for PCR and usually works anyway).

Good luck!

The blood was freshly drawn on the day of isolation. But anyways, thanks for that answer

krystle on Nov 23 2009, 11:09 AM said:

Hi, I used to do blood extraction during my internship but I couldn't recall my exact protocol. I answer based on my memory...
I assume your protocol is like this:
First, wash with PBS, spin down remove supernatant, add lyse buffer and after your lyse cells, do you spin down the cells and remove the red supernatant before add proteinase K?
Try Increase your volume of P:C:I. Make sure you are getting the top clear layer and discard the rest.
Hope this help.

Adrian

I use the MinElute PCR purification kit from Qiagen. You can clean several times if necessary. Then I add lots of BSA during the PCR. Good luck.