6 replies to this topic

[thegene]

Dear mates,

The first thing I want to ask for is that one of my colleagues has used the salt method to purify DNA from blood, and the resulted DNA was not in good concentration and contained some brownish color (blood contamination). I was thinking to use phenol to repurify them. Anybody knows any way to repurify These samples since they already have a part of the DNA already extracted.

Secondly, i will really appretiate it if anybody can provide me with the standard protocol for purifying DNA from blood using phenol.



Thank you in advance

[ksturm]

Why not removing erythrocytes prior DNA-extraction (density gradient centrifugation)? Could solve problem of brown color.
Purification by Phenol-Chloroform-extraction: nucleicacidsolution is extracted with one volume phenol (pH 8), After that with One Volume of phenol-Chloroform-isoamylethanol (25:24:1 v/v), After that with One volume chloroform. In between centrifugate and take topmost aqueous phase (without interphase) to new vessel. Proteins are denaturated and accumulate in organic phase, DNA stays in aqueous phase. Final Preciptation with ethanol will remove phenol. Don't use polystyrol or polycarbonat made vessels when centrifuge. They cant stay organic solvents. Phenol is toxic! Beware of Vapours.

Edited by ksturm, 21 October 2010 - 07:34 AM.

[thegene]

Hi,
Thank you so much for your reply!
regarding the brown color in the blood sample; If i understood what you mean is that just centrifuging the tube to get rid of the blood right?
Do not you think that if i centrifuge the tubes i will get the blood and the DNA in the bottom so it will be hard to seperate them isnt it?

[ksturm]

As I understand, you did already a purification of DNA from blood sample and this DNA solution is contaminated. repurify this DNA solution as I described above. This will remove proteins. probably brown color dues to contamination with haem. This will be hard to remove. Haem will inhibit PCR reaction.
Next time doing a DNA purification from blood, think about eliminating erythrocytes. They barely contain DNA, so PBMC may sufficient for your expermient. You can get them by density gradient centrifugation with ficoll. actually there are very good kits for DNA preparation from blood (pharmacia, qiagen, promega).

Edited by ksturm, 21 October 2010 - 07:32 AM.

[Maddie]

Qiagen has many kits to extract and/or purify DNA from blood. You need to get rid of the hematin that inhibits the PCR.

Edited by Maddie, 21 October 2010 - 08:57 AM.

[Kikuyu]

The Qiagen Gentra Kit is great for DNA extraction from blood. Just make sure that you start with an RBC lysis step (we use 1xlysis buffer, then the Qiagen RBC lysis solution)

[newborn]

Dont you want to idolate cell-free DNA? If not, the hemolysis step is the key for your method. Make sure your cells in the blood completelly suspended not aggregated before hemolytic process.